Agomelatine attenuates cisplatin-induced cognitive impairment via modulation of BDNF/TrkB signaling in rat hippocampus.

Cisplatin is a drug used effectively in the treatment of malignant tumors. However, cisplatin has many side effects, including cognitive impairment. Agomelatine, a synthetic melatonin analogue, is an important antidepressant. Increasing evidence has shown that agomelatine may be a potential neuroprotective agent. The aim of this study was to investigate the effect of agomelatine on learning and memory functions in cisplatin-induced cognitive impairment in a rat model. Male rats were administered agomelatine and cisplatin for 4 weeks. Neurobehavioral tests were performed at the end of the 4th week. After behavioral tests, rats were euthanized and BDNF, TNF, IL-1ß, MDA and GSH levels were measured in hippocampal homegenates by ELISA. In addition, nNOS and TrkB receptor activity were measured immunohistochemically. The results showed that agomelatine significantly improved cognitive functions in spatial memory tests in rats with cisplatin-induced cognitive impairment. In addition, agomelatine treatment positively affected the discrimination index (DI). On the other hand, agomelatine treatment elevated cisplatin-suppressed hippocampal BDNF levels. Agomelatine treatment reduced cisplatin-induced neuroinflammation by suppressing TNF and IL-1ß levels. Similarly, agomelatine reduced oxidative stress in the hippocampus. Histological findings showed that agomelatine treatment reduced pyramidal neuron damage in hippocampal DG, CA1 and CA3. Cisplatin increased nNOS and TrkB positivity in DG, CA1 and CA3 neurons compared to control. In contrast, agomelatine treatment decreased both nNOS and TrkB positive scores. These findings indicate that agomelatine reduces cisplatin-related cognitive impairment by exerting anti-inflammatory action and possibly by the modulation of the BDNF/TrkB/nNOS pathways in the hippocampus.

Identification of the potential matrix protein of invertebrate iridescent virus 6 (IIV6).

Invertebrate iridescent virus 6 (IIV6) is a nucleocytoplasmic virus with a ∼212 kb linear dsDNA genome that encodes 215 putative open reading frames (ORFs). Proteomic analysis has revealed that the IIV6 virion consists of 54 virally encoded proteins. Interactions among the structural proteins were investigated using the yeast two-hybrid system, revealing that the protein of 415R ORF interacts reciprocally with the potential envelope protein 118L and the major capsid protein 274L. This result suggests that 415R might be a matrix protein that plays a role as a bridge between the capsid and the envelope proteins. To elucidate the function of 415R protein, we determined the localization of 415R in IIV6 structure and analyzed the properties of 415R-silenced IIV6. Specific antibodies produced against 415R protein were used to determine the location of the 415R protein in the virion structure. Both western blot hybridization and immunogold electron microscopy analyses showed that the 415R protein was found in virions treated with Triton X-100, which degrades the viral envelope. The 415R gene was silenced by the RNA interference (RNAi) technique. We used gene-specific dsRNA's to target 415R and showed that this treatment resulted in a significant drop in virus titer. Silencing 415R with dsRNA also reduced the transcription levels of other viral genes. These results provide important data on the role and location of IIV6 415R protein in the virion structure. Additionally, these results may also shed light on the identification of the homologs of 415R among the vertebrate iridoviruses.

COVID-19 vaccine immunity in oncology patients.

BACKGROUND: To investigate the effect of vaccine types applied in our country against 2019 coronavirus disease on the formation of protective antibodies in oncology patients. MATERIALS AND METHODS: The data of 81 cancer patients who received at least one dose of vaccine for COVID-19 and radiotherapy were analyzed retrospectively. At any time after the vaccination, blood samples were taken and the antibody titers against the vaccine were measured. RESULTS: There were 28 (34.6 %) patients who received two doses of vaccine and 48 patients (59.3 %) who received 3 doses of vaccine (Sinovac only), while 26 patients (32.1 %) were given both vaccines. The mean time for antibody measurement was 62 days after the last vaccination. IgG levels were significantly higher in patients who received Biontech vaccine than in those who received Sinovac (r = 0.525; p < 0.001). While chemotherapy was the factor that decreased the mean IgM level (p = 0.044), advanced disease (stages 3 and 4) was a significant factor that increased the mean IgG level (p = 0.047). A statistically significant negative correlation was found between IgM antibody level and WBC count after first vaccination (r = ‒0.251; p = 0.024). For every WBC count unit increase in the first vaccination period, there was a 1.333-fold increase in the risk of IgM negativity. CONCLUSION: The Biontech vaccine produced higher antibody levels in advanced oncology patients. While the application of radiotherapy in cancer patients was not found to be an effective factor in the vaccination status, it was determined that the application of chemotherapy significantly reduced IgM levels (Tab. 5, Ref. 28). Text in PDF www.elis.sk Keywords: COVID-19 pandemic, COVID-19 vaccine, cancer patients, radiotherapy, chemotherapy, SARS-CoV-2 IgM and IgG, abscopal effect.

Inhibition of methotrexate induced toxicity in the adult rat spleen by adalimumab.

Methotrexate (MTX) has been in use for the treatment of rheumatoid arthritis (RA), psoriasis, and cancer since 1948. Its toxic side effects on tissues and organs have been well documented but splenotoxicity has not been addressed. This study set out to investigate this issue by examining the effectiveness of anti-TNFα agents against MTX-induced toxicity in T lymphocytes and macrophages via the regulation of CD3, CD68, and CD200R. Twenty-four Sprague Dawley rats were allocated to three groups: control (received saline solution only), MTX (20 mg/kg of single-dose of MTX), and Ada + MTX (single dose of 10 mg/kg Adalimumab before MTX administration). The spleens were removed 5 days after MTX administration. The number of CD3+/mm3cells for the control, MTX and Ada + MTX groups were, respectively, 2.69 ± 0.86, 20.51 ± 2.7, (p = 0.000) and 11.07 ± 2.01 (p = 0.000). The number of CD68+ macrophages/mm3 in the control, MTX and Ada + MTX groups were, respectively, 8.62 ± 1.08, 38.19 ± 1.37 (p = 0.000), and 16.87 ± 12.57 (p = 0.000). The number of macrophages that were CD200R+/mm3 in the control, MTX, and Ada + MTX groups were 3.33 ± 1.66, 25.77 ± 2.37 (p = 0.000), and 8.68 ± 2.66 (p = 0.000), respectively. We also observed that Ada reduced the numerical densities of these cells following MTX administration (p < 0.05). Ada may, therefore, be a promising candidate for the prevention of the deleterious effects on T lymphocytes and macrophages of MTX-induced toxicity.

Effects of gadodiamide and gadoteric acid on lung tissue: A comparative study.

We set out to investigate the effects of gadodiamide and gadoteric acid, used for magnetic resonance imaging, on the lungs. In this study, 32 male Sprague Dawley rats were used. These were allocated into four groups; The first group (control) was untreated. The second group received isotonic saline on the first and fourth days of the week for 5 weeks. Following the same schedule, the third and fourth groups received a total of 2 mg/kg gadodiamide and gadoteric acid, respectively, in place of saline. The alveolar Wall thickness was evaluated. Gadodiamide and gadoteric acid significantly increased the numbers of collagen-3 and caspase-3 positive cells in the lung tissue (p < 0.05). In addition, these two substances increased the alveolar Wall thickness (p < 0.05). Furthermore, they increased the levels of malondialdehyde and glutathione (p < 0.05). This study demonstrates that both linear and macrocyclic contrast agents are toxic for the lungs in rats.

Effect of the prenatal electromagnetic field exposure on cochlear nucleus neurons and oligodendrocytes in rats.

Electromagnetic radiation from elecromagnetic field (EMF) sources has been an important health concern for a long time. The vast majority of this exposure is due to the widespread use of mobile phones, an important source of the EMF. The EMF generated by mobile phones may have adverse effects on the various biological structures that regulate the body system and function. In this study, it was aimed to evaluate histopathologically the effects of 900-megahertz (MHz) EMF application in the prenatal period on the development of the ventral cochlear nucleus, which is the first place of hearing in the brainstem, at various time points of the postnatal period in rats. In the study, Sprague-Dawley pregnant rats were divided randomly into two groups as the control group and the EMF group. The rats in the EMF group were exposed to a 900-MHz EMF every day until birth, while no EMF was applied to the rats in the control group. Auditory brainstem responses of both groups were recorded on the postnatal 13th day, the day the hearing starts. Newborn rats were sacrificed by anesthesia on days 7, 10, 15, and 30. Contrary to the control group, structural damage in cochlear nuclear neurons and oligodendrocyte cell structures and increased caspase-3 activity were observed in the postnatal period in the EMF groups. However, no significant difference was observed between the groups in terms of structural damage and caspase-3 activity at different stages of the postnatal period when cochlear nucleus development was observed. According to ABS, there was no significant difference between the average latency of waves in both groups. In conclusion, this study shows that 900-MHz electromagnetic waves propagated from mobile phones during the prenatal period have no harmful effects on the development of the ventral cochlear nucleus of rats.

Apelin-13 activates the hippocampal BDNF/TrkB signaling pathway and suppresses neuroinflammation in male rats with cisplatin-induced cognitive dysfunction.

It has been established that cisplatin causes neuronal damage and cognitive impairment. However, the mechanism is not sufficiently clear. Apelin-13 is an endogenous peptide with strong neuroprotective effects through the synthesis of neurotrophic factors and suppression of inflammation. The aim of this study was to investigate the role of brain-derived neurotrophic factor/tropomyosin receptor kinase B (BDNF/TrkB) signaling pathway and the potential inhibitory effects of apelin-13 in the mechanism of cisplatin-induced hippocampal damage and cognitive impairment. Apelin-13 was administered to adult sprague dawley male rats at a dose of 20 nmol/kg every day for 4 weeks, cisplatin was administered at a dose of 5 mg/kg once a week for 4 weeks. The spatial and recognition memory tests of the rats were performed on the 5th week. BDNF and the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) levels were measured by ELISA in hippocampal homogenates. Pyramidal neuron and glial cell damage in the hippocampal CA1, CA3 and dentate gyrus (DG) were analyzed histologically. TrkB activity in the hippocampus was determined by immunohistochemical methods. Cisplatin impaired spatial and recognition memory in rats, while apelin-13 improved spatial memory but did not affect recognition memory. Cisplatin suppressed BDNF in the hippocampus while increased IL-1ß and TNF-α. In contrast, apelin-13 administration increased BDNF but significantly suppressed TNF-α and IL-1B. Cisplatin caused pyramidal neuron and glial cell damage in CA1, CA3 and DG. In the cisplatin + apelin-13 group, however, pyramidal neuron and glial cell damage was less than those without apelin-13. Cisplatin increased TrkB activity in the hippocampus, which was counteracted by apelin-13. In conclusion, apelin-13 reduced the cisplatin-induced cognitive deficiency, by suppressing inflammation and stimulating the synthesis and activation of neurotrophic factors in hippocampal tissue.

Glass particle contamination threat in nursing practice: A pilot study.

AIM: The aim of this study was to determine the level of glass particle contamination from medical ampoules during breakage in nursing practice and their removal by filtration. DESIGN: Glass medical ampoules were broken-open as instructed and contamination was assessed microscopically. METHODS: Three types of medial ampoules (A, B, C) were used. Dispensation of contents was carried out using 21 or 22 G needles, with and without syringe-filters. Particles were determined by light microscopy. This study was conducted between April 2018 and January 2020. RESULTS: Glass particles of 0.94-90.70 µm were detected in 94% of all samples. There were 48, 162 and 201 glass particles in groups, A, B and C, respectively. Filtration had no effect in group A but was effective up to 85% in the other groups. CONCLUSION: This study confirms that ampoule contents are contaminated with glass particles during the opening procedure, which varies with make and content. Syringe-filter use can be up to 85% effective in their removal. IMPACT: Use of syringe-filter can decrease glass particle contamination up to 85%. Innovative nursing studies are needed to minimize or completely avoid particule contamination.

Correlation of Suicidal Thoughts and Toxoplasmosis in Patients With Depression.

Objective We investigated the correlation between serum anti-Toxplasma gondii IgG and suicidal thoughts in depressive patients. Methods Depressive patients with (n = 100) and without (n = 100) suicidal thoughts along with 100 healthy control subjects were recruited for this study. In all three groups, a semi-structured clinical interview form called Structured Clinical Interview for DSM-IV (Diagnostic and Statistical Manual of Mental Disorders) Axis-I Disorder (SCID-I), Hamilton Depression Rating Scale (HAMD), suicidal behavior scale, and a sociodemographic data form were completed. Sera from all participants were taken, and anti-toxoplasma IgG was measured by Enzyme Linked Immunosorbent Assay (ELISA)-Chemiluminescent Microparticle Immunoassay. Statistical analysis of the data was performed. Results The serum anti-toxoplasma IgG levels of patients with suicidal thoughts were significantly higher than those without suicidal thoughts and the controls, which were 80.04 ± 40.66, 78 ± 14.82, and 19.98 ± 14.65, respectively, p < 0.001. There was no correlation between toxoplasma IgG and HAMD score in patients lacking suicidal thoughts (r = -0.112, p = 0.463). Conclusion This study shows a correlation between seropositivity for anti-Toxoplasma gondii IgG and depression with suicidal thoughts.

Prenatal Effects of a 1,800-MHz Electromagnetic Field on Rat Livers.

The use of devices, including mobile phones, generating electromagnetic fields (EMF) is widespread and is progressively increasing. It has also been shown that EMF may have detrimental effects. This is the first study to investigate the postnatal biochemical and histological effects of prenatal exposure of rat livers to 1,800-MHz EMF at different time intervals in uteroplacental life. The 3 EMF groups of rats were exposed to 1,800-MHz EMF for 6, 12, or 24 h daily for 20 days. Unexposed rats served as control group. All rats were subjected to anesthesia, and on postnatal day 60, the livers were excised, and blood was collected for histological and biochemical analyses. Malondialdehyde levels were significantly higher in the exposed groups than the unexposed controls (p < 0.05). In contrast, EMF-exposed groups had lower liver tissue glutathione levels than controls (p < 0.05). Serum Ca2+, alanine transaminase, and aspartate aminotransferase levels were higher in EMF-exposed groups than controls (p < 0.05). In addition, liver tissue total oxidant status levels were increased (p < 0.05), and liver tissue total antioxidant status levels were decreased (p < 0.05) compared to the control group. Furthermore, in the EMF groups, extensive vacuolation and degeneration of the hepatocytes in the portal area, as well as those surrounding the sinusoids, were evident. Affected hepatocytes had polygonally shaped nuclei and vacuolic cytoplasm imparting eosinophilic staining. Loss of cellular membrane integrity and invaginations, as well as picnotic nuclei, was prominent. This study has shown that intrauterine liver damage caused by 1,800-MHz EMF exposure persists into puberty in rats.

The effects of mobile phone exposure on mast cells in rat dura mater / Efectos de la exposición del teléfono móvil en los mastocitos de la duramadre de ratas

Mobile phone use has increased rapidly. The central nervous system has been shown to be adversely affected by its electromagnetic field (EMF) resulting in headache and sleep disturbances. How the cells make up the CNS and are affected by EMF is unclear. However, because of their central role in inflammation through diverse stimuli including radiation, this study aimed to investigate the effects of electromagnetic fields induced by mobile phones on mast cells in rat dura mater. A total of 18 adult, female, SpragueDawley rats were divided into two groups. The choice of female rats for his study was based on recent surveys demonstrating that mobile phone use is more frequent and prolonged among females. The study group was exposed to 900 MHz electromagnetic field (1 h/day for 45 days). In the end of the study, duramater tissue was extracted and stained using Toluidine blue. Mast cells were counted and results were analysed using Student t test. Mean mast cell number was 202.33±9.82 and 456.78±35.01 in the control and study groups, respectively (p<0.05). Analysis of serum electrolyte and immunoglobulin E levels showed no statistically significant difference between the two groups (p>0.05). The study showed that mobile phone exposure increased mast cell number and degranulation in rat dura mater. Further studies are required to evaluate the clinical implications of these findings.

El uso del teléfono móvil ha aumentado rápidamente. Se ha demostrado que el sistema nervioso central (SNC) se ve afectado de manera adversa debido al campo electromagnético (CEM) que produce dolor de cabeza y trastornos del sueño. No está claro cómo se ve afectada la composición celular del SNC por el CEM. Sin embargo, debido a su función principal en la inflamación a través de diversos estímulos que incluyen la radiación, este estudio tuvo como objetivo investigar los efectos de los campos electromagnéticos inducidos por los teléfonos móviles en los mastocitos de la duramadre de ratas. Un total de 18 ratas Sprague-Dawley adultas, hembras, se dividieron en dos grupos. Se usaron ratas hembras para este estudio en base a investigaciones recientes que han demostrado que el uso de teléfonos móviles es más frecuente y prolongado en las mujeres. Los grupos de estudio fueron expuestos a un campo electromagnético de 900 MHz (1 h / día durante 45 días). Al término del estudio, fue extirpado el tejido de la duramadre y teñido con azul de toluidina. Se contaron los mastocitos y se analizaron los resultados utilizando la prueba t de Student. La cantidad media de células cebadas fue de 202,33 ± 9.82 y 456,78 ± 35,01 en los grupos control y estudio, respectivamente (p <0,05). El análisis del electrolito sérico y los niveles de inmunoglobulina E no mostraron diferencias estadísticamente significativas entre los dos grupos (p> 0,05). El estudio mostró que la exposición a teléfonos móviles aumentó el número de mastocitos y la desgranulación en la duramadre de las ratas. Se requieren estudios adicionales para evaluar las implicaciones clínicas de estos hallazgos.

Protective effects of amifostine, curcumin and caffeic acid phenethyl ester against cisplatin-induced testis tissue damage in rats.

Cisplatin is an effective antineoplastic drug that is usually used to treat a number of different types of cancer in the clinic. One of the most notable side effects of cisplatin use is infertility. The present study was designed to determine the non-oxidative testicular effects caused by the use of cisplatin in rats. The rats were randomly allocated to the experimental groups. The untreated rats represented the control group (group I) and the treatment groups were as follows: cisplatin alone (group II), cisplatin+amifostine (group III), cisplatin+curcumin (group IV), and cisplatin+caffeic acid phenethyl ester (CAPE; group V). The present study observed that following cisplatin administration, the expression of nuclear factor-κB (NF-κß)/p65, caspase-3 and 8-deoxyguanosine (8-OHdG) increased in germinal epithelium and Leydig cells. However, the expression of these markers decreased in groups III-V, most notably in the group treated with amifostine. cisplatin induced-damage was countered by amifostine and curcumin. The results revealed that the activation of NF-κB, caspase-3 and 8-OHdG had a significant role in cisplatin-induced testicular toxicity. Thus, amifostine, curcumin and, to a lesser extent, CAPE have the potential for use as therapeutic adjuvants in cisplatin-induced testis injury.

Evaluation of the effects of fasting associated dehydration on maternal NGAL levels and fetal renal artery Doppler parameters.

OBJECTIVE: The aim of this study was to evaluate maternal neutrophil gelatinase-asssociated lipocalin (NGAL) levels and fetal renal artery (fRA) Doppler flow indices in pregnant women fasting in Ramadan in respect of dehydration in long hot summer days as a marker of hypoperfusion and early renal injury. METHODS: A cross-sectional observational study was carried out at a University Hospital. Fasting pregnant women and non-fasting age, gravidity and gestational age-matched women were evaluated for hematologic, blood biochemistry and urine parameters in the first and fourth weeks of the Ramadan. Umbilical artery and fRA Doppler flows were studied in each evaluation. RESULTS: Blood urea nitrogen, potassium and hematocrit levels, blood and urine NGAL levels were significantly higher, and fRA Doppler indices increased in fasting women (p < 0.05) during the second visit in the last week of the Ramadan, while non-fasting women had no significant alterations in each evaluation (p > 0.05). CONCLUSIONS: Adequate maternal vascular volume is essential for the maintenance of healthy pregnancy. Fasting during the long and hot summer days leads to fluid deprivation and dehydration which was found to be related to subclinical maternal renal dysfunction and increased fRA Doppler indices.

Marine-Lenhart syndrome with papillary thyroid carcinoma.

Graves' disease with accompanying functioning nodules is known as Marine-Lenhart syndrome. Autonomously functioning thyroid nodules (AFTNs) also within Graves' thyroid tissue are almost always bening in nature. A 45-year-old man developed hyperthyroidism due to the coexistence of Graves' disease and AFTN. Total thyroidectomy was performed. The hyperfunctioning nodule with centrally hypoactive foci detected by technetium-99m thyroid scanning was histologically diagnosed as papillary thyroid carcinoma that was 2.5 cm in diameter. We report the presence of papillary thyroid carcinoma within AFTN in patients with Marine-Lenhart syndrome, which has not been reported so far.

Contribution of neutrophil activation in the differentiation of urine infection and contamination in pregnant women.

BACKGROUND: Urinary tract infections are among the most common bacterial infections of humans. Urine culture is the gold standard for asymptomatic bacteriuria and pyuria is not always present in bacteriuria, nor is it specific for bacteriuria. OBJECTIVE: The aim of the present study was to determine neutrophil activation and the contributions of this activation in the differentiation of infection and contamination. METHODS: The serum and urine myeloperoxidase (MPO) levels of 50 pregnant females with symptoms suggesting UTI and 25 healthy non-pregnant control subjects were measured using the enzyme-linked immunosorbent assay (ELISA) method and the obtained values were compared with the results of urine microscopy and urine culture. RESULTS: The leukocyte count in urine was significantly higher in group 1 (infection) and group 2 (contamination) when compared with the control group (group 1 mean: 18.2; group 2 mean: 14.2; control mean: 4.8; ANOVA test, p ≤ 0.00). According to the obtained ELISA values, a statistical difference in the levels of urine MPO between the patient and control groups was seen (p ≤ 0.00). There was no statistical difference among the groups for serum MPO levels (p ≥ 0.451). CONCLUSION: The study findings suggest that standardized measurement techniques such as dipstick screening assay for urine MPO level may be useful in differentiating infection and contamination, especially in pregnant patients.

Wernicke's encephalopathy in a patient with unresectable gastric carcinoma and literature review.

Wernicke's encephalopathy (WE) is a disease classically associated with nutrition deficiency. It is characterized by typical symptoms like confusion, ataxia and ophthalmoparesis, and developes due to thiamine deficieny in alcoholic patients. Recently, it has been shown that WE could ocur in patients with gastric carcinoma without a history of alcohol use. In this paper, we have made some suggestions about early diagnosis, treatment and prevention of WE by discussing the development of WE in a patient with unresectable gastric carcinoma, who had been inpatient for a long time and given radiotherapy and chemotherapy.

Lymphocyte subpopulations in Sheehan's syndrome.

The role of autoimmunity in the development of Sheehan's syndrome is obscure. There are a limited number of studies investigating the immunological alterations accompanying Sheehan's Syndrome. Our objective was to evaluate lymphocyte subsets in these patients. We conducted a cross-sectional clinical study. Cytofluorometry was used for the immunophenotyping of peripheral blood leukocytes from patients with Sheehan's syndrome followed up in the endocrine clinic during 2005-2009. Fifteen consecutive patients (mean age 61.6 ± 11.3, range 34-75 years) and 25 healthy controls (mean age 56.7 ± 10.6, range 34-80 years) were included. There was no statistically significant difference between the groups in terms of mean age. The percentages of CD19(+), CD16(+)/56(+), CD8(+)28(-), γδTCR(+), CD8(+); the total lymphocyte counts; and the ratio of CD8(+)28(-)/CD8(+)28(+) were similar (p > 0.05) between patients and controls. Whereas the leucocyte counts (p = 0.003), the percentage of CD3 (+) DR (+) (p < 0.001), CD8(+)28(+) (p = 0.030), CD4(+)CD25(+) (p = 0.007), the ratio of CD3 (+) DR(+)/CD3 (p < 0.001) were higher; the percentage of CD3 (p = 0.020), CD4 (p < 0.001) and the ratio of CD4/CD8 (p = 0.006) were lower in patients with Sheehan's syndrome compared to healthy controls. There was a positive correlation between the duration of illness and the percentage of CD3(+)DR(+) (r = 0.53, p = 0.03) expression. Some peripheral lymphocyte cell subsets show marked variation in patients with Sheehan's syndrome in comparison to matched healthy subjects, which may have implications for altered immune regulation in these patients. High CD3 (+) DR (+) expression that correlates with the duration of illness in Sheehan's patients is suggestive of an ongoing inflammation accompanying the slow progression of pituitary dysfunction in Sheehan's syndrome. It is not clear if these cellular alterations contribute to the cause or consequence of pituitary deficiency in Sheehan's syndrome.

IL-2 overcomes the unresponsiveness but fails to reverse the regulatory function of antigen-induced T regulatory cells.

Intranasal administration of peptide Ac1-9[4Y], based on the N-terminal epitope of myelin basic protein, can induce CD4(+) T cell tolerance, and suppress experimental autoimmune encephalomyelitis induction. The peptide-induced regulatory T (PI-T(Reg)) cells failed to produce IL-2, but expressed IL-10 in response to Ag and could suppress naive T cell responses in vitro. Analysis of Jak-STAT signaling pathways revealed that the activation of Jak1, STAT3, and STAT5 were induced in tolerant T cells after Ag stimulation in vivo. In addition, the expression of suppressor of cytokine signaling 3 was induced in tolerant T cells, suggesting that cytokines regulate the tolerant state of the PI-T(Reg) cells. Stimulation of PI-T(Reg) cells in vitro with IL-10 induced Jak1 and STAT3 activation, but not STAT5, suggesting that IL-10 is important, but not the only cytokine involved in the development of T cell tolerance. Although IL-2 expression was deficient, stimulation with IL-2 in vitro induced Jak1 and STAT5 activation in PI-T(Reg) cells, restored their proliferative response to antigenic stimulation, and abrogated PI-T(Reg)-mediated suppression in vitro. However, the addition of IL-2 could not suppress IL-10 expression, and the IL-2 gene remained inactive. After withdrawal of IL-2, the PI-T(Reg) cells regained their nonproliferative state and suppressive ability. These results underline the ability of the immune system to maintain tolerance to autoantigens, but at the same time having the ability to overcome the suppressive phenotype of tolerant T cells by cytokines, such as IL-2, during the protective immune response to infection.

Regulation of Raf-1 activation and signalling by dephosphorylation.

The Raf-1 kinase is regulated by phosphorylation, and Ser259 has been identified as an inhibitory phosphorylation site. Here we show that the dephosphorylation of Ser259 is an essential part of the Raf-1 activation process, and further reveal the molecular role of Ser259. The fraction of Raf-1 that is phosphorylated on Ser259 is refractory to mitogenic stimulation. Mutating Ser259 elevates kinase activity because of enhanced binding to Ras and constitutive membrane recruitment. This facilitates the phosphorylation of an activating site, Ser338. The mutation of Ser259 also increases the functional coupling to MEK, augmenting the efficiency of MEK activation. Our results suggest that Ser259 regulates the coupling of Raf-1 to upstream activators as well as to its downstream substrate MEK, thus determining the pool of Raf-1 that is competent for signalling. They also suggest a new model for Raf-1 activation where the release of repression through Ser259 dephosphorylation is the pivotal step.