Tissue-based T cell activation and viral RNA persist for up to 2 years after SARS-CoV-2 infection.

The mechanisms of postacute medical conditions and unexplained symptoms after SARS-CoV-2 infection [Long Covid (LC)] are incompletely understood. There is growing evidence that viral persistence, immune dysregulation, and T cell dysfunction may play major roles. We performed whole-body positron emission tomography imaging in a well-characterized cohort of 24 participants at time points ranging from 27 to 910 days after acute SARS-CoV-2 infection using the radiopharmaceutical agent [18F]F-AraG, a selective tracer that allows for anatomical quantitation of activated T lymphocytes. Tracer uptake in the postacute COVID-19 group, which included those with and without continuing symptoms, was higher compared with prepandemic controls in many regions, including the brain stem, spinal cord, bone marrow, nasopharyngeal and hilar lymphoid tissue, cardiopulmonary tissues, and gut wall. T cell activation in the spinal cord and gut wall was associated with the presence of LC symptoms. In addition, tracer uptake in lung tissue was higher in those with persistent pulmonary symptoms specifically. Increased T cell activation in these tissues was also observed in many individuals without LC. Given the high [18F]F-AraG uptake detected in the gut, we obtained colorectal tissue for in situ hybridization of SARS-CoV-2 RNA and immunohistochemical studies in a subset of five participants with LC symptoms. We identified intracellular SARS-CoV-2 single-stranded spike protein-encoding RNA in rectosigmoid lamina propria tissue in all five participants and double-stranded spike protein-encoding RNA in three participants up to 676 days after initial COVID-19, suggesting that tissue viral persistence could be associated with long-term immunologic perturbations.

An Enhanced Role of Innate Immunity in the Immune Response After Kidney Transplant in People Living With HIV: A Transcriptomic Analysis.

BACKGROUND: Although kidney transplantation (KT) has become the standard of care for people living with HIV (PLWH) suffering from renal failure, early experiences revealed unanticipated higher rejection rates than those observed in HIV- recipients. The cause of increased acute rejection (AR) in PLWH was assessed by performing a transcriptomic analysis of biopsy specimens, comparing HIV+ to HIV- recipients. METHODS: An analysis of 68 (34 HIV+, 34 HIV-) formalin-fixed paraffin-embedded (FFPE) renal biopsies matched for degree of inflammation was performed from KT recipients with acute T cell-mediated rejection (aTCMR), borderline for aTCMR (BL), and normal findings. Gene expression was measured using the NanoString platform on a custom gene panel to assess differential gene expression (DE) and pathway analysis (PA). RESULTS: DE analysis revealed multiple genes with significantly increased expression in the HIV+ cohort in aTCMR and BL relative to the HIV- cohort. PA of these genes showed enrichment of various inflammatory pathways, particularly innate immune pathways associated with Toll-like receptors. CONCLUSIONS: Upregulation of the innate immune pathways in the biopsies of PLWH with aTCMR and BL is suggestive of a unique immune response that may stem from immune dysregulation related to HIV infection. These findings suggest that these unique HIV-driven pathways may in part be contributory to the increased incidence of allograft rejection after renal transplantation in PLWH.

The Cocktail Effects on the Acute Cytotoxicity of Pesticides and Pharmaceuticals Frequently Detected in the Environment.

Xenobiotics never appear as single, isolated substances in the environment but instead as multi-component mixtures. However, our understanding of the ecotoxicology of mixtures is far from sufficient. In this study, three active pharmaceutical ingredients (carbamazepine, diclofenac, and ibuprofen) and three pesticides (S-metolachlor, terbuthylazine, and tebuconazole) from the most frequently detected emerging micropollutants were examined for their acute cytotoxicity, both individually and in combination, by bioluminescence inhibition in Aliivibrio fischeri (NRRL B-11177). Synergy, additive effects, and antagonism on cytotoxicity were determined using the combination index (CI) method. Additionally, PERMANOVA was performed to reveal the roles of these chemicals in binary, ternary, quaternary, quinary, and senary mixtures influencing the joint effects. Statistical analysis revealed a synergistic effect of diclofenac and carbamazepine, both individually and in combination within the mixtures. Diclofenac also exhibited synergy with S-metolachlor and when mixed with ibuprofen and S-metolachlor. S-metolachlor, whether alone or paired with ibuprofen or diclofenac, increased the toxicity at lower effective concentrations in the mixtures. Non-toxic terbuthylazine showed great toxicity-enhancing ability, especially at low concentrations. Several combinations displayed synergistic effects at environmentally relevant concentrations. The application of PERMANOVA was proven to be unique and successful in determining the roles of compounds in synergistic, additive, and antagonistic effects in mixtures at different effective concentrations.

In vivo estrogenicity of glyphosate, its formulations, and AMPA on transgenic zebrafish (Danio rerio) embryos.

In this study, the disrupting effects of glyphosate (GLY), aminomethylphosphonic acid (AMPA), and three glyphosate-based herbicides (GBHs) on vitellogenesis in a non-concentration-dependent manner are reported for the first time in 120 h of acute exposure of zebrafish at environmentally relevant concentrations. GBHs are commonly used worldwide in weed control management. Due to their extensive application, they frequently occur in aquatic ecosystems and may affect various organisms. The active substance GLY and its major by-product, AMPA, are the most thoroughly studied chemicals; however, the adverse effects of the complex formulas of GBHs with diverse and unknown content of co-formulants are still not sufficiently researched. This study focused on the embryotoxicity, sublethal malformations, and estrogenic potency of GLY, AMPA, and four commonly used GBHs on zebrafish embryos using a wild type and an estrogen-sensitive, transgenic zebrafish line (Tg(vtg1:mCherry)). After 120 h of exposition, AMPA did not cause acute toxicity, while the LC50 of GLY was 160 mg/L. The GBHs were more toxic with LC50 values ranging from 31 to 111 GLY active equivalent (a.e.) mg/L. Exposure to 0.35-2.8 mg/L GBHs led to sublethal abnormalities: typical symptoms were structural deformation of the lower jaw and anomalies in the olfactory region. Deformity rates were 10-30% in the treated groups. In vivo, fluorescently expressed vtg1 mCherry protein in embryonic liver was detected by a non-invasive microscopic method indicating estrogenic action through vitellogenin production by GLY, AMPA, and GBHs. To confirm the in vivo findings, RT-qPCR method was performed to determine the levels of the estrogenicity-related vtg1 mRNA. After 120 h of exposure to GLY, AMPA, and three GBHs at a concentration of 0.35 mg/L, the expression of vtg1 gene was significantly up-regulated. Our results highlight the risk that short-term GLY and GBH exposure can cause developmental malformations and disrupt the hormonal balance in zebrafish embryos.

Plumage color degradation indicates reproductive effort: an experiment.

Plumage color has traditionally been regarded as a static ornamental trait, but evidence is accumulating for significant color changes without molt that typically reduce the conspicuousness of ornamentation. In some species, the social partner seems to increase its reproductive investment if the color trait is experimentally enhanced, suggesting that color change could act as a signal. However, the information content of this signal is so far unclear. For example, birds in poor condition or making greater effort may deteriorate more severely. We used brood size manipulations to alter the reproductive effort of male and female collared flycatchers Ficedula albicollis. Both sexes showed less severe decline in some reflectance attribute of their white breast when their brood was experimentally reduced. In each sex, greater deterioration of the reflectance trait affected by the manipulation was accompanied by increased feeding rate by the partner. These feeding patterns do not prove, but are consistent with, a compensatory response by the partner to induced degradation. The manipulation effects on color change we detected confirm for the first time that plumage color deterioration can indicate current reproductive effort, thereby providing a potential fitness advantage to social partners that react to such deterioration.

Multimodal Molecular Imaging Reveals Tissue-Based T Cell Activation and Viral RNA Persistence for Up to 2 Years Following COVID-19.

The etiologic mechanisms of post-acute medical morbidities and unexplained symptoms (Long COVID) following SARS-CoV-2 infection are incompletely understood. There is growing evidence that viral persistence and immune dysregulation may play a major role. We performed whole-body positron emission tomography (PET) imaging in a cohort of 24 participants at time points ranging from 27 to 910 days following acute SARS-CoV-2 infection using a novel radiopharmaceutical agent, [18F]F-AraG, a highly selective tracer that allows for anatomical quantitation of activated T lymphocytes. Tracer uptake in the post-acute COVID group, which included those with and without Long COVID symptoms, was significantly higher compared to pre-pandemic controls in many anatomical regions, including the brain stem, spinal cord, bone marrow, nasopharyngeal and hilar lymphoid tissue, cardiopulmonary tissues, and gut wall. Although T cell activation tended to be higher in participants imaged closer to the time of the acute illness, tracer uptake was increased in participants imaged up to 2.5 years following SARS-CoV-2 infection. We observed that T cell activation in spinal cord and gut wall was associated with the presence of Long COVID symptoms. In addition, tracer uptake in lung tissue was higher in those with persistent pulmonary symptoms. Notably, increased T cell activation in these tissues was also observed in many individuals without Long COVID. Given the high [18F]F-AraG uptake detected in the gut, we obtained colorectal tissue for in situ hybridization SARS-CoV-2 RNA and immunohistochemical studies in a subset of participants with Long COVID symptoms. We identified cellular SARS-CoV-2 RNA in rectosigmoid lamina propria tissue in all these participants, ranging from 158 to 676 days following initial COVID-19 illness, suggesting that tissue viral persistence could be associated with long-term immunological perturbations.

Kisspeptin-8 Induces Anxiety-Like Behavior and Hypolocomotion by Activating the HPA Axis and Increasing GABA Release in the Nucleus Accumbens in Rats.

Kisspeptins (Kp) are RF-amide neuropeptide regulators of the reproductive axis that also influence anxiety, locomotion, and metabolism. We aimed to investigate the effects of intracerebroventricular Kp-8 (an N-terminally truncated octapeptide) treatment in Wistar rats. Elevated plus maze (EPM), computerized open field (OF), and marble burying (MB) tests were performed for the assessment of behavior. Serum LH and corticosterone levels were determined to assess kisspeptin1 receptor (Kiss1r) activation and hypothalamic-pituitary-adrenal axis (HPA) stimulation, respectively. GABA release from the nucleus accumbens (NAc) and dopamine release from the ventral tegmental area (VTA) and NAc were measured via ex vivo superfusion. Kp-8 decreased open arm time and entries in EPM, and also raised corticosterone concentration, pointing to an anxiogenic effect. Moreover, the decrease in arm entries in EPM, the delayed increase in immobility accompanied by reduced ambulatory activity in OF, and the reduction in interactions with marbles show that Kp-8 suppressed exploratory and spontaneous locomotion. The increase in GABA release from the NAc might be in the background of hypolocomotion by inhibiting the VTA-NAc dopaminergic circuitry. As Kp-8 raised LH concentration, it could activate Kiss1r and stimulate the reproductive axis. As Kiss1r is associated with hyperlocomotion, it is more likely that neuropeptide FF receptor activation is involved in the suppression of locomotor activity.

Use of the Tissue Common Rejection Module Score in Kidney Transplant as an Objective Measure of Allograft Inflammation.

Long-term kidney transplant (KT) allograft outcomes have not improved as expected despite a better understanding of rejection and improved immunosuppression. Previous work had validated a computed rejection score, the tissue common rejection module (tCRM), measured by amplification-based assessment of 11 genes from formalin-fixed paraffin-embedded (FFPE) biopsy specimens, which allows for quantitative, unbiased assessment of immune injury. We applied tCRM in a prospective trial of 124 KT recipients, and contrasted assessment by tCRM and histology reads from 2 independent pathologists on protocol and cause biopsies post-transplant. Four 10-µm shaves from FFPE biopsy specimens were used for RNA extraction and amplification by qPCR of the 11 tCRM genes, from which the tCRM score was calculated. Biopsy diagnoses of either acute rejection (AR) or borderline rejection (BL) were considered to have inflammation present, while stable biopsies had no inflammation. Of the 77 biopsies that were read by both pathologists, a total of 40 mismatches in the diagnosis were present. The median tCRM scores for AR, BL, and stable diagnoses were 4.87, 1.85, and 1.27, respectively, with an overall significant difference among all histologic groups (Kruskal-Wallis, p < 0.0001). There were significant differences in tCRM scores between pathologists both finding inflammation vs. disagreement (p = 0.003), and both finding inflammation vs. both finding no inflammation (p < 0.001), along with overall significance between all scores (Kruskal-Wallis, p < 0.001). A logistic regression model predicting graft inflammation using various clinical predictor variables and tCRM revealed the tCRM score as the only significant predictor of graft inflammation (OR: 1.90, 95% CI: 1.40-2.68, p < 0.0001). Accurate, quantitative, and unbiased assessment of rejection of the clinical sample is critical. Given the discrepant diagnoses between pathologists on the same samples, individuals could utilize the tCRM score as a tiebreaker in unclear situations. We propose that the tCRM quantitative score can provide unbiased quantification of graft inflammation, and its rapid evaluation by PCR on the FFPE shave can become a critical adjunct to help drive clinical decision making and immunosuppression delivery.

Further Evidence That the Soluble Urokinase Plasminogen Activator Receptor Does Not Directly Injure Mice or Human Podocytes.

BACKGROUND: The role of the soluble urokinase plasminogen activator receptor (suPAR) in focal segmental glomerulosclerosis (FSGS) as the circulating factor or as a predictor of recurrence after transplantation remains controversial. Previously published studies in mice and isolated podocytes produced conflicting results on the effect of suPAR on podocyte injury, effacement of foot processes, and proteinuria. These discordant results were in part due to diverse experimental designs and different strains of mice. The aim of our study was to determine the reasons for the inconsistencies of the previous studies results with suPAR by using uniform methods and studies in different strains of mice. METHODS: We utilized a primary culture of human podocytes and 2 mouse models, the wild type (WT) and the urokinase plasminogen activator receptor (uPAR) KO (uPAR), in an attempt to resolve the reported conflicting results. RESULTS: In both WT and uPAR mouse models, injection of recombinant uPAR, even at a high dose (100 µg), did not induce proteinuria, effacement of podocytes, or disruption of the cytoskeleton. Injection of suPAR resulted in its deposition exclusively in the glomerular endothelial cells and not in the podocytes of WT mice and was not detected at the uPAR KO mice. Kidneys from patients with recurrent FSGS had negative immunostaining for uPAR. We also evaluated the effect of recombinant uPAR on primary culture of human podocytes. uPAR did not result in podocytes damage. CONCLUSIONS: suPAR by itself is not the cause for direct podocyte injury, in vitro or in vivo. These findings suggest a more complex and still poorly understood role of suPAR in FSGS.

Changes in striatal dopamine release and locomotor activity following acute withdrawal from chronic nicotine are mediated by CRF1, but not CRF2, receptors.

The aim of the present study was to investigate the participation of corticotropin-releasing factor (CRF) receptors (CRF1 and CRF2) in the alterations of the dorsal and ventral striatal dopamine release and the vertical and horizontal locomotor activity observed in rats following chronic nicotine treatment and consequent acute withdrawal. In this purpose, male Wistar rats were exposed to repeated intraperitoneal (ip) injection with nicotine or saline solution for 7 days. On the 8th day or the 9th day the rats were injected intracerebroventricularly (icv) with selective CRF1 antagonist antalarmin or selective CRF2 antagonist astressin2B or saline solution. Thirty minutes after the icv injection the changes of the horizontal and vertical locomotor activity were recorded in an in vivo conducta system. Immediately after the behavioral recordings the changes of the dorsal and ventral striatal dopamine release were determined in an in vitro superfusion system. On the 8th day, the horizontal and vertical locomotor activities and the dorsal and ventral striatal dopamine releases increased significantly in nicotine-treated rats, compared to the saline-treated ones. On the 9th day, the horizontal locomotor activity and the dorsal striatal dopamine release increased significantly, whereas the vertical locomotor activity and the ventral striatal dopamine release decreased significantly in nicotine-treated rats, compared to the saline-treated ones. All the changes observed were attenuated significantly by antalarmin, but not astressin2B. The present study demonstrates that the changes of striatal dopamine release and locomotor activity observed following chronic nicotine treatment and consequent acute withdrawal are mediated by CRF1, but not CRF2, receptor.

Identifying a potential biomarker for primary focal segmental glomerulosclerosis and its association with recurrence after transplantation.

BACKGROUND: We investigated circulating levels of individual soluble urokinase plasminogen activation receptor (suPAR) forms to determine if specific circulating fragments of suPAR (II-III) and (I) can better serve as clinical biomarkers for focal segmental glomerulosclerosis (FSGS) and the risk of recurrence after transplantation. MATERIALS AND METHODS: Serum levels of intact suPAR and its cleaved forms were measured with two assays, ELISA and TR-FIA. RESULTS: suPAR levels in healthy controls were significantly lower than those who had glomerular diseases but were not significantly different between FSGS patients and glomerular controls. Intact suPAR (I-II-III) levels were noted to be elevated in glomerular diseases including FSGS. uPAR fragment (I) levels measured with the TR-FIA 4 assay were significantly higher in FSGS (695.4 + 91.29 pMol/L) than glomerular controls (239.1 + 40.45 pMol/L, P = 0.001). However, suPAR(I) levels were not significantly different between recurrent FSGS and nonrecurrent FSGS patients. CONCLUSION: Our analysis of suPAR using the ELISA assay used in all previous studies does not appear to be a useful marker for FSGS nor serve as a predictor for its recurrence after transplantation. The TR-FIA assay results suggest that uPAR(I) is a potential biomarker for FSGS but not of its recurrence.

The effects of the urocortins on the hypothalamic-pituitary-adrenal axis - similarities and discordancies between rats and mice.

The urocortins (Ucn I, Ucn II and Ucn III) are structural analogues of corticotropin-releasing factor (CRF). The aim of our present experiments was to compare the effects of the urocortins on the hypothalamic-pituitary-adrenal (HPA) axis in rats and mice, including the hypothalamic adrenocorticotropic hormone (ACTH) secretagogues, such as CRF and arginine vasopressin (AVP). Therefore, male CFLP mice and male Wistar rats were injected intracerebroventricularly (icv) with 0.5, 1, 2 and 5 µg/2 µl of Ucn I, Ucn II or Ucn III. After 30 min the animals were decapitated, and then, hypothalamic CRF and AVP concentrations and plasma ACTH and corticosterone (CORT) levels were measured. All measurements were performed by enzyme-linked immunosorbent assays (ELISA), except that of the plasma CORT level, which was determined by chemofluorescent assay. Ucn I increased significantly the hypothalamic CRF and AVP concentrations in both rats and mice. Ucn II and Ucn III influenced significantly only the hypothalamic CRF concentration in rats, without affecting the hypothalamic AVP concentration. In contrast, Ucn II and Ucn III increased significantly only the hypothalamic AVP concentration in mice, without affecting the hypothalamic CRF concentration. The hypothalamic changes were reflected more or less accurately by changes of the plasma ACTH and CORT levels. The present experiments demonstrate that the urocortins regulate the HPA axis centrally via modulation of the hypothalamic ACTH secretagogues and that there are some similarities and discordancies between rats and mice regarding this regulation.

Kisspeptin modulates pain sensitivity of CFLP mice.

Kisspeptin, a hypothalamic neuropeptide, is a member of the RF-amide family, which have been known to modify pain sensitivity in rodents. The aim of the present study was to investigate the effect of kisspeptin-13 (KP-13), an endogenous derivative of kisspeptin, on nociception in adult male and female CFLP mice and the possible interaction of KP-13 with morphine on nociception. Mice were injected with different doses of KP-13, 30, 60 and 120 min after of which the nociceptive sensitivity were assessed via the tail-flick test. To investigate the receptor involved in the mediation a kisspeptin receptor antagonist (KP-234) pretreatment was applied before KP-13 administration. Furthermore, we investigated the effect of KP-13 on the acute antinociceptive effect of morphine, on acute morphine tolerance and on naloxone-precipitated withdrawal. Last, the Von Frey test was used in order to assess KP-13's effect on mechanical nociception. Our results showed that KP-13 decreased the nociceptive threshold of both males and females independent of sex, which was prevented by KP-234. Furthermore, KP-13 treatment depressed the acute antinociceptive effect of morphine and attenuated the development of morphine tolerance. KP-13 also induced a mechanical hypersensitivity. These data underlie kisspeptin's hyperalgesic action and argues for the role of kisspeptin receptor 1 in the mediation of its action. Furthermore, our results suggest that central KP-13 administration can modify the acute effects of morphine.

Anxiolytic- and antidepressant-like actions of Urocortin 2 and its fragments in mice.

The aim of the present study was to investigate the potential anxiolytic- and antidepressant-like actions of Urocortin 2 (Ucn2) and its two fragments, Ucn2 (1-21) and Ucn2 (22-38), in mice, in an attempt to identify the biologically active sequence of this 38 amino acid neuropeptide. In this purpose, male C57BL/6 mice were treated intracerebroventricularly (icv) with 0.125, 0.25, 0.5 and 1 µg/2 µl of Ucn2, Ucn2 (1-21) or Ucn2 (22-38). After 30 min, the mice were evaluated in an elevated plus-maze test and a forced swim test for anxiety- and depression-like behavior, respectively. Each test lasted 5 min. Ucn2 at dose of 0.25 µg/2 µl and Ucn2 (1-21) at dose of 0.125 µg/2 µl, but not Ucn2 (22-38), increased significantly the number of entries into and the time spent in the open-arms, without influencing the total number of entries. In parallel, the same doses of Ucn2 and Ucn2 (1-21), but not Ucn2 (22-38), increased significantly the climbing and the swimming activity, while decreasing significantly the time of immobility. In addition, Ucn2 at doses of 0.125 µg/2 µl and 0.5 µg/2 µl decreased significantly the time of immobility, but they did not change the other parameters. The present study demonstrates that Ucn2 exerts anxiolytic- and antidepressant-like effects in C57BL/6 mice, which are mediated by the N-terminal, but not the C-terminal fragment of the peptide. The establishment of the smallest active sequence by further fragmentation of Ucn2 (1-21) may allow the synthesis of new anxiolytic and antidepressant drugs.

Accuracy of the hypothetical sky-polarimetric Viking navigation versus sky conditions: revealing solar elevations and cloudinesses favourable for this navigation method.

According to Thorkild Ramskou's theory proposed in 1967, under overcast and foggy skies, Viking seafarers might have used skylight polarization analysed with special crystals called sunstones to determine the position of the invisible Sun. After finding the occluded Sun with sunstones, its elevation angle had to be measured and its shadow had to be projected onto the horizontal surface of a sun compass. According to Ramskou's theory, these sunstones might have been birefringent calcite or dichroic cordierite or tourmaline crystals working as polarizers. It has frequently been claimed that this method might have been suitable for navigation even in cloudy weather. This hypothesis has been accepted and frequently cited for decades without any experimental support. In this work, we determined the accuracy of this hypothetical sky-polarimetric Viking navigation for 1080 different sky situations characterized by solar elevation θ and cloudiness ρ, the sky polarization patterns of which were measured by full-sky imaging polarimetry. We used the earlier measured uncertainty functions of the navigation steps 1, 2 and 3 for calcite, cordierite and tourmaline sunstone crystals, respectively, and the newly measured uncertainty function of step 4 presented here. As a result, we revealed the meteorological conditions under which Vikings could have used this hypothetical navigation method. We determined the solar elevations at which the navigation uncertainties are minimal at summer solstice and spring equinox for all three sunstone types. On average, calcite sunstone ensures a more accurate sky-polarimetric navigation than tourmaline and cordierite. However, in some special cases (generally at 35° ≤ θ ≤ 40°, 1 okta ≤ ρ ≤ 6 oktas for summer solstice, and at 20° ≤ θ ≤ 25°, 0 okta ≤ ρ ≤ 4 oktas for spring equinox), the use of tourmaline and cordierite results in smaller navigation uncertainties than that of calcite. Generally, under clear or less cloudy skies, the sky-polarimetric navigation is more accurate, but at low solar elevations its accuracy remains relatively large even at high cloudiness. For a given ρ, the absolute value of averaged peak North uncertainties dramatically decreases with increasing θ until the sign (±) change of these uncertainties. For a given θ, this absolute value can either decrease or increase with increasing ρ. The most advantageous sky situations for this navigation method are at summer solstice when the solar elevation and cloudiness are 35° ≤ θ ≤ 40° and 2 oktas ≤ ρ ≤ 3 oktas.

Effects of kisspeptin on diabetic rat platelets.

Hyperglycemia, hyperlipidemia, and free radicals result in platelet activation and atherogenesis. Kisspeptin (KP) is able to regulate metabolism, hemostasis, and the development of atherosclerosis. We examined whether platelet aggregation of streptozotocin-induced diabetic rats depends on the inducer type and if KP-13 and RF-9 (a kisspeptin receptor modifier) can influence platelet function. We measured the speed and the maximum of aggregation, along with the area under the curve. Serum glucose and calcium levels and urine formation of diabetic animals increased, while the body mass and platelet count decreased. Collagen was the most effective inducer of platelet aggregation. The aggregability of nondiabetic platelets was elevated in the presence of 5 × 10-8 mol/L KP-13. This effect was less expressed in diabetic animals. The effectivity of RF-9 was stronger than that of KP-13 in nondiabetic platelets, however it was ineffective in diabetic animals. RF-9 pre-treatment did not change the effects of 5 × 10-8 mol/L KP-13 in either animal group. The in vivo activation of diabetic platelets, which may be due to elevated serum calcium, induces thrombocytopenia and may lead to reduced in vitro aggregability. We could not demonstrate the antagonistic effect of RF-9 against KP-13 in isolated platelets.

In Vitro Characterization of Human Mesenchymal Stem Cells Isolated from Different Tissues with a Potential to Promote Complex Bone Regeneration.

Bone tissue regeneration is a major, worldwide medical need, and several strategies have been developed to support the regeneration of extensive bone defects, including stem cell based bone grafts. In addition to the application of stem cells with high osteogenic potential, it is important to maintain proper blood flow in a bone graft to avoid inner graft necrosis. Mesenchymal stem cells (MSCs) may form both osteocytes and endothelial cells; therefore we examined the combined in vitro osteogenic and endothelial differentiation capacities of MSCs derived from adipose tissue, Wharton's jelly, and periodontal ligament. Based on a detailed characterization presented here, MSCs isolated from adipose tissue and periodontal ligament may be most appropriate for generating vascularized bone grafts.

Selective CRF2 receptor agonists ameliorate the anxiety- and depression-like state developed during chronic nicotine treatment and consequent acute withdrawal in mice.

The aim of the present study was to investigate the effects of the selective agonists of the corticotropin-releasing factor (CRF) 2 receptor, urocortin 2 (UCN 2) and urocortin 3 (UCN 3), on the anxiety- and depression-like signs induced by acute nicotine withdrawal in mice. In order to do so, male CFLP mice were exposed for 7 days to repeated intraperitoneal (IP) injection with nicotine or saline solution and 1day of acute withdrawal and then a single intracerebroventricular (ICV) injection with UCN 2, UCN 3 or saline solution. After 30min the mice were observed in an elevated plus-maze test or a forced swim test, for anxiety- and depression-like behavior. After 5min of testing, the plasma corticosterone concentration reflecting the activity of the hypothalamic-pituitary-adrenal (HPA) axis was also determined by a chemo-fluorescent method. Half of the animals were treated ICV and evaluated on the 8th day, the other half on the 9th day. On the 8th day, nicotine-treated mice presented signs of anxiolysis and depression, but no significant elevation of the plasma corticosterone concentration. On the 9th day, nicotine-treated mice exhibited signs of anxiety and depression and a significant increase of the plasma corticosterone levels. Central administration of UCN 2 or UCN 3 ameliorated the anxiety- and depression-like state including the hyperactivity of the HPA axis, developed during acute withdrawal following chronic nicotine treatment. The present study suggests that selective CRF2 receptor agonists could be used as a therapy in nicotine addiction.

Longitudinal measurement invariance in prospective oral health-related quality of life assessment.

BACKGROUND: Prospective assessments of oral health-related quality of life (OHRQoL) changes are prone to response shift effects when patients reconceptualize, reprioritize, or recalibrate the perceived meanings of OHRQoL test items. If this occurs, OHRQoL measurements are not "invariant" and may reflect changes in problem profiles or perceptions of OHRQoL test items. This suggests that response shift effects must be measured and controlled to achieve valid prospective OHRQoL measurement. The aim of this study was to quantify response shift effects of Oral Health Impact Profile (OHIP) scores in prospective studies of prosthodontic patients. METHODS: Data came from the Dimensions of Oral Health-Related Quality of Life Project. The final sample included 554 patients who completed the OHIP questionnaire on two occasions: pre- and post-treatment. Only items that compose the 14-item OHIP were analyzed. Structural equation models that included pre- and post-treatment latent factors of OHRQoL with different across-occasion constraints for factor loadings, intercepts, and residual variances were fit to the data using confirmatory factor analysis. RESULTS: Data fit both the unconstrained model (RMSEA = .038, SRMR = .051, CFI = .92, TLI = .91) and the partially constrained model with freed residual variances (RMSEA = .037, SRMR = .064, CFI = .92, TLI = .92) well, meaning that the data are well approximated by a one-factor model at each occasion, and suggesting strong factorial across-occasion measurement invariance. CONCLUSIONS: The results provided cogent evidence for the absence of response shift in single factor OHIP models, indicating that longitudinal OHIP assessments of OHRQoL measure similar constructs across occasions.

The effect of kisspeptin on the regulation of vascular tone.

Kisspeptin has been implicated in cardiovascular control. Eicosanoids play a crucial role in the activation of platelets and the regulation of vascular tone. In the present study, we investigated the effect of kisspeptins on eicosanoid synthesis in platelets and aorta in vitro. Platelets and aorta were isolated from Wistar-Kyoto rats. After preincubation with different doses of kisspeptin, samples were incubated with [1-(14)C]arachidonic acid (0.172 pmol/mL) in tissue culture Medium 199. The amount of labeled eicosanoids was measured with liquid scintillation, after separation with overpressure thin-layer chromatography. Kisspeptin-13 stimulated the thromboxane synthesis. The dose-response curve was bell-shaped and the most effective concentration was 2.5 × 10(-8) mol/L, inducing a 27% increase. Lipoxygenase products of platelets displayed a dose-dependent elevation up to the dose of 5 × 10(-8) mol/L. In the aorta, kisspeptin-13 induced a marked elevation in the production of 6-keto-prostaglandin F1α, the stable metabolite of prostacyclin, and lipoxygenase products. Different effects of kisspeptin on cyclooxygenase and lipoxygenase products indicate that beyond intracellular Ca(2+) mobilization, other signaling pathways might also contribute to its actions. Our data suggest that kisspeptin, through the alteration of eicosanoid synthesis in platelets and aorta, may play a physiologic and (or) pathologic role in the regulation of vascular tone.