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These results could be verified in the B16-EpCAM model, although the absolute survival rates cannot be compared in the two models. Therapy with either the trAb BiLu or the anti-CTLA-4 mAb HB304 alone yielded identical survival benefits in comparison to the tumor control group. This effect could be improved by combining both therapy regimens resulting in a prolongation of the median survival and an increase of long-term survivors from 20 to 40% (Figure 3C).
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study
| 100.0 |
Taken together, a moderate bonus effect on direct tumor killing may be achieved by using the combination approach. Since trAbs not only mediate direct tumor cell killing, but also elicit a vaccination effect , we then asked whether the immunologic memory induced by trAbs is affected by the combination with anti-CTLA-4 treatment. These experiments were only done using the B78-D14 melanoma because the B16-EpCAM model is less stringent due to its higher inherent immunogenicity (our unpublished data). Since effective long-lasting tumor protection is dependent on T-cell responses of the Th1 type [27, 28], we first compared the levels of Th1 and Th2 cytokines in sera of mice that had been vaccinated with trAb Surek or HB304 or the antibody combination together with replication-incompetent tumor cells as an antigen source. After Surek immunization, high concentrations of Th1 cytokines were detected (Figure 4A). The data confirmed our previous results, which indicated a clear Th1 bias involving IL-12 expression by DCs and IFN-γ production by T cells . Accordingly, an upregulation of Th2 cytokines was also observed, because this is a prerequisite for inducing Th1 responses (see Discussion). In contrast, HB304 alone did not induce any cytokine response. Furthermore, the cytokine levels that were induced by Surek could not be further enhanced by combining trAb treatment with HB304 (Figure 4A). Likewise, when T cells from vaccinated mice were restimulated with melanoma-derived peptides in vitro, the combination group neither showed enhanced IFN-γ release as compared to animals vaccinated with Surek alone (not shown).
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study
| 100.0 |
Immunization was done as outlined in Materials and Methods using the indicated immunogens. Irradiated B78-D14 cells as an antigen source were included in each setting. (A) Th1/Th2 cytokines in sera of mice on day 21 after starting immunization. Up to 8 mice were included in each group. For all cytokines tested, the difference between the Surek and the Surek/HB304 group was not significant, while these two groups were different from all other groups with P < 0.005 (Mann-Whitney). (B) Tumor-directed antibodies in sera of mice vaccinated with different immunogens. Binding of serum IgG1 and IgG2a antibodies to melanoma cells was measured by flow cytometry. Mean fluorescence intensities (MFI) are shown as a quantitative measure for antibody coating of tumor cells. Up to 5 animals were included in each group. The columns show means and SEM. Significances (Mann-Whitney) are indicated. The differences between IgG1 titers are not significant. (C) On day 21, mice were challenged with a lethal dose of melanoma cells. The significance is denoted (logrank).
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study
| 100.0 |
Since it is established that trAb-dependent vaccination induces tumor-specific humoral immune responses , which correlate with tumor protection in vivo, we then measured the titers of melanoma-directed antibodies in sera of vaccinated mice. While specific antibodies of the IgG1 subclass were not significantly affected by any vaccination protocol, immunization with Surek induced elevated levels of the IgG2a subclass (Figure 4B). HB304 induced anti-melanoma antibody titers that were not significantly different from untreated controls. However, combining Surek with HB304 strongly enhanced the IgG2a induction effected by Surek monotherapy (Figure 4B).
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study
| 100.0 |
As the humoral IgG2a response is a predictive parameter for the immunologic antitumor memory , we then compared Surek and Surek/HB304 vaccination with regard to melanoma rejection in vivo. Challenge with a lethal dose of viable melanoma cells three weeks after start of the immunization showed that the vaccination effect induced by Surek is indeed significantly enhanced when the trAb is combined with HB304 during the immunization phase. While 40% of mice survived in the Surek-immunized group, about 80% were effectively protected when both antibodies were used for vaccination (Figure 4C). Thus, the bonus effect of the combination approach appeared far more pronounced in terms of anti-tumor memory than direct tumor cell killing.
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study
| 100.0 |
Having shown that the combinatorial vaccination improves the humoral IgG2a antitumor response and the long-lasting protection against melanoma as compared to trAb immunization alone, we analyzed in more detail the T-cell compartment in differentially immunized mice. To discriminate effector, effector-memory and central-memory cells, T cells isolated from immunized mice and restimulated in vitro were analyzed for expression of CD44 and CD62L (Figure 5A). It turned out that the fraction of memory CD4+ T cells was markedly increased in the group having undergone the combinatorial vaccination (Figure 5B).
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study
| 100.0 |
T cells from spleens of differentially vaccinated mice were stimulated in vitro with splenocytes that were loaded with melanoma-derived peptides . After 7 days, T cells were analyzed for expression of CD4, CD8, CD44 and CD62L. At least 4 mice were included in each group. (A) Representative diagram delineating the differentiation between different memory and effector T-cell compartments. (B) Quantitation of CD4+ T-cell subpopulations from differentially vaccinated mice. (C) Expression of CD137 on CD4+ T cells after in vivo immunization and restimulation with melanoma peptides. CD137+ cells were exclusively identified as CD44+ memory cells (not shown). (D) In the CD8+ T-cell population, an expansion of the memory compartment or a significant upregulation of CD137 was not induced by the combination approach. In all panels, columns represent mean values and SEM. Significances are also shown (Mann-Whitney).
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study
| 100.0 |
CD137 is a costimulatory molecule that is expressed on the surface of T cells upon antigen-specific and TCR-dependent stimulation. Interestingly, CD137 was elevated on CD4+ T cells that had been primed in vivo by irradiated tumor cells and Surek together with HB304 (Figure 5C). These CD137+ cells were exclusively identified as CD44+ memory cells (data not shown). By contrast, neither an expansion of the memory compartment nor a significant upregulation of CD137 was seen in the CD8+ T-cell population (Figure 5D).
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study
| 100.0 |
Combinatorial approaches of cancer immunotherapy have attracted much interest in the past years. Specifically, the simultaneous blockade of different immune checkpoints has been extensively evaluated in translational and clinical studies [8–10]. The rationale is to release the brake imposed to bona fide tumor-reactive T cells by surface molecules like CTLA-4, whose induction is considered as a counter-regulatory mechanism required for self-limitation of any immune response [29, 30]. However, tumor escape may be more effectively counteracted if reagents with different modes of action are combined. Therefore, we nourished the blockade of CTLA-4 with trAbs that specifically redirect T cells to TAAs and activate the T cells in the presence of tumor cells. Thus, the unspecific effect of anti-CTLA-4 is supplemented with an antigen-specific component because after trAb treatment, specifically activated T cells with induced CTLA-4 expression can be further stimulated by anti-CTLA-4 mAbs. As a consequence, the redirection of activated T cells by trAbs could be beneficial for the side-effect profile of checkpoint inhibitors. This is indicated by experiments using an allogeneic transplantation setting, which also inherently bears the danger of tissue damage by uncontrolled T-cell activity. Here, T cells could be redirected away from healthy tissues to the tumor site by trAbs, thereby significantly reducing graft-versus-host-disease .
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study
| 99.94 |
In other studies, the antitumor efficacy of bispecific antibody constructs cross-linking HER2 or CD33 with CD3 could also be improved by blocking PD-L1, which is expressed on tumor cells and inhibits T-cell responses via ligation of PD-1 [32, 33]. In contrast to these constructs, trAbs have a unique Fc region comprising the mouse IgG2a and the rat IgG2b isotype, which enables the preferential interaction with activating Fc receptors . Thus, trAbs not only mediate direct cell lysis but also induce a long-lasting T cell-dependent immunologic antitumor memory, because accessory cells expressing activating Fc receptors are stimulated and present tumor-derived peptides to the immune system [13, 15–17]. The vaccination effect exerted by trAbs involves a polyvalent T-cell response, which is directed against numerous TAAs and does not depend on the antigen targeted by the therapeutic trAb [16, 17]. This was shown by challenging mice with the parental melanoma cell line B16F0, which did not express the antigens recognized by the trAb used for vaccination. Polyvalent immune responses are thought to be superior to immunity against single TAAs because tumor immune escape due to antigen loss becomes less likely [34, 35].
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study
| 99.94 |
A major aim of our study was therefore to examine the trAb-induced vaccination effect in combination with CTLA-4 blockade. Importantly, while direct trAb-mediated destruction of tumor cells was only moderately improved by combination with CTLA-4 inhibition, the immunologic memory elicited by the combination treatment was indeed considerably enhanced. The memory induced was associated with an expansion of CD4+ memory cells, whose CD137 expression indicated preceding antigen-specific activation (Figure 5). These phenotypic changes were not seen in the CD8+ T-cell compartment.
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study
| 100.0 |
Because a similar increase of Th1 cytokines was seen in all trAb-vaccinated groups (Figure 4A), the combination approach had no impact on the Th1 response, which is instrumental for effective tumor protection [27, 28]. The simultaneous upregulation of Th2 cytokines in all groups where Th1 cytokines were induced was not unexpected since Th2 cytokines are required for priming Th1 responses [34, 36, 37]. A clear hierarchy, however, was detected for the humoral immune response induced by the different antibodies (Figure 4B). Thus, the improved immunologic memory observed after vaccination with trAbs combined with CTLA-4 blockade correlated with increased serum titers of melanoma-reactive IgG2a antibodies. Indeed, humoral immunity has been shown to be relevant for tumor protection in vivo . The finding of CD4+ rather than CD8+ memory cells after combined vaccination (Figure 5) supports this concept.
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study
| 100.0 |
It is well known that CTLA-4 blocking increases Treg proliferation . Accordingly, the proliferation and the amounts of Tregs were enhanced after combinatorial vaccination. Importantly, this increased Treg proliferation was not responsible for the expansion of the antigen-experienced memory CD4+ T cells (manuscript in preparation), as might be predicted by a study using another tumor model where Tregs primarily contributed to the CD137+CD44+CD4+ memory compartment .
|
study
| 100.0 |
Thus, the improved immunization effect did not depend on a reduction of Treg numbers, but possibly (at least partly) on a functional inhibition of Tregs. As it has been shown that Tregs control B-cell expression of CD80 and CD86 via the coreceptor CTLA-4 , a potential mechanism enhancing the vaccination effect of trAbs in the combination setting may be mediated by CTLA-4 blockade on Tregs, thus fostering the induction of antitumor antibodies (Figure 4B). Further mechanistic studies, which should also address the role of follicular helper cells, are warranted.
|
study
| 100.0 |
Up to now, checkpoint inhibitors have been most effective in melanoma, bladder cancer and lung cancer. Other types of malignant disease where checkpoint inhibitors still have to prove their effectiveness should also be addressed by combining with trAb immunization. Therefore, the positive effect of blocking CTLA-4 during trAb vaccination on the CD4+ T cell-associated antitumor memory argues for further evaluation of this concept in preclinical and clinical settings.
|
study
| 54.6 |
TrAbs were produced by quadroma technology as described [16, 25]. Quadroma-derived supernatants were purified by protein A chromatography applying sequential pH elution followed by a cationic exchange chromatography purification step. Surek [17, 19, 25, 26] is a trAb that was generated by fusion of the parental hybridomas 17A2 (anti-mouse CD3, rat IgG2b) and Me361 (anti-GD2, mouse IgG2a) . BiLu comprises the same anti-CD3 specificity and additionally includes a mouse IgG2a binding arm recognizing human EpCAM, which was derived from the clone C215 . For production of HB304 (anti-mouse CTLA-4), the hamster hybridoma clone UC10-4F10-11 was used. Cell lines were cultivated in chemically defined protein-free medium. All antibodies were manufactured by Trion Research GmbH.
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study
| 100.0 |
B78-D14 and B16-EpCAM cells were grown in RPMI 1640 medium supplemented with 8.9% and 5%, respectively, fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1× nonessential amino acids. Further, 400 μg/ml G418 and 500 μg/ml Hygromycin B were added to B78-D14. Cells were extensively washed in PBS before application. The identity of the cell lines was regularly confirmed on the basis of cell morphology and the expression of the transgenes.
|
study
| 99.94 |
C57BL/6 mice were purchased from Taconic (Ry, Denmark). All animal experiments were performed at least twice with 5 to 10 female animals included in each group. For testing trAb-induced tumor rejection, mice received a challenge of 105 B78-D14 or B16-EpCAM cells and were treated on day 2 and 5 with 50 μg Surek or 10 μg BiLu. 100 μg HB304 were either given simultaneously with trAbs or, in some experiments, repeatedly on days 9 and 12 followed by four other injections in weekly intervals.
|
study
| 99.94 |
To examine the long-term immunologic memory, mice were immunized on day 0 and 14 with 105 irradiated B78-D14 cells (100 Gy) together with 10 μg Surek. Other groups were additionally treated with 100 μg HB304 on days 4, 8, 13 and 20. On day 21, all mice received a challenge of 3 × 103 viable B16F0 melanoma cells.
|
study
| 100.0 |
All cells and antibodies were delivered i.p. except for B16-EpCAM cells that were i.v. administered. Control groups receiving tumor cells and PBS only were included in each experiment. Mice were euthanized when signs of tumor growth became visible. All animal experiments were in accordance with animal welfare regulations and had been approved by the competent authority.
|
other
| 99.9 |
T-cell analyses were performed by fluorescence-activated cell sorting (FACS) using an LSRII or a FACS Calibur flow cytometer and the cell quest analysis program (BD Bioscience, Heidelberg, Germany). T-cell surface markers were directly stained with fluorescence-labeled mAbs against CD4 (clone RM4-5; BD Biosciences), CD8 (53-6.7, eBioscience, San Diego, USA), CD44 (IM7; BD Biosciences), CD62L (MEL-14; eBioscience) and CD69 (H1.2F3; BD Bioscience). Ki67 (SolA15; eBioscience), CTLA-4 (UC10-4B9; BioLegend, or HB304) and the transcription factor FoxP3 (FJK-16s; eBioscience) were stained intracellularly.
|
study
| 99.94 |
Immature DCs were prepared by culturing bone marrow precursors from C57BL/6 mice in RPMI 1640 supplemented with 20% FCS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin, 50 μM 2-mercaptoethanol, sodium pyruvate and nonessential amino acids in the presence of 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF). Medium was replaced every second day, cells were frozen at -140°C on day 7. Frozen DCs were thawed, counted and directly applied to T-cell stimulation assays.
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study
| 100.0 |
4 × 106 T-cells, which were isolated from spleens of naïve C57BL/6 mice by panning of B lymphocytes with anti-IgG+M antibodies (Dianova, Hamburg, Germany), were co-cultivated with 2 × 105 DCs and 105 irradiated (100 Gy) tumor cells in 24-well plates for three days. TrAbs were added at 1 μg/ml. Cells were cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-Glutamine, 1 mM sodium pyruvate, 1× non-essential amino acids, 10 mM HEPES and 50 μM 2-mercaptoethanol. CD69 and CTLA-4 surface expression of CD4+ and CD8+ T cells was determined by flow cytometry at 0, 24, 48 and 72 h using the mAbs listed above. The percentage of positive cells was determined in comparison to corresponding isotype controls.
|
study
| 100.0 |
In-vitro stimulation of T cells was conducted by co-culturing 5 × 106 splenocytes of immunized mice with 5 × 106 splenocytes of naïve mice, which were loaded with Trp-2, MAGE-A5 and gp100 peptides and irradiated at 30 Gy, in the presence of IL-2 (30 U/ml; Amersham Pharmacia, Freiburg, Germany) in 24-well plates. After 7 days, T cells were harvested and characterized by FACS analysis as described above.
|
study
| 99.94 |
To determine IFN-γ release of activated T cells, 5 × 105 splenocytes of immunized mice were co-cultured with 5 × 105 peptide-loaded splenocytes of naïve mice in 96-well plates. Supernatants were collected 24 h and 48 h later to measure IFN-γ concentrations.
|
study
| 100.0 |
IFN-γ in supernatants was quantitated by enzyme-linked immunosorbent assay (Ready-SET-go IFN-γ ELISA Kit; eBioscience) according to the manufacturer´s instructions. Th1/Th2 cytokines in sera of mice were analyzed by using the Bio-PlexTM cytokine assay (Bio-Rad, München, Germany).
|
study
| 99.94 |
Tumor-specific antibodies were analyzed in sera after immunization and tumor challenge as described above. Sera were taken about 3 weeks following the last trAb injection. At this time point, no residual trAb was detected. Tumor cells were pre-incubated with sera on ice; after washing cells, fluorescence-labeled antibody against mouse IgG1 or IgG2a was added. Analysis of IgG-binding tumor cells was subsequently performed using FACS (LSRII flow cytometer; BD Biosciences).
|
study
| 100.0 |
HIV and AIDS continue to be a major public health problem where almost 70 million people have been infected with the Human Immune deficiency Virus and about 35 million people have died of AIDS since the beginning of the epidemic . Globally, there are 3.3 million children under fifteen years living with HIV infection whose 95% of them acquired the infection through Mother to Child Transmission (MTCT) . Without intervention the rate of MTCT cumulatively from pregnancy to cessation of breastfeeding ranges from 25 to 48% [2–4]. Prevention of Mother To Child Transmission (PMTCT) program focuses on HIV pre and post test counseling, feeding option and use of Highly Active antiretroviral Therapy (HAART) during pregnancy, intrapartum and postpartum. Studies has shown that use of Anti Retro Viral drugs (ARV) can significantly reduce MTCT of HIV to less than 2% . The 2012 WHO guideline recommends that countries with limited laboratory and infrastructure capacity to provide universal access to CD4 cell count testing, all confirmed HIV-infected pregnant and breastfeeding women must be offered life¬long triple ART as soon as diagnosed regardless of their CD4 count or clinical stage as part of the new Option B+ . Success in preventing MTCT will not only depend on use of antiretroviral prophylaxis, but also on continuing support to the nursing mothers from their male partners. It has been shown that male partner involvement reduces the risk of vertical transmission and infant mortality by 40% compared to non-involvement . Mechanism by which male involvement contributes to the effectiveness of PMTCT include male socio cultural superiority and influence in many of the Africa settings . Women learn and retain more information when educated together with their partners and some pregnant mothers don't accept HIV testing till they have their partners consent or assent . Indeed utilization of PMTCT services by pregnant women is affected by several factors such as fear of disclosure of HIV results, lack of male partner support, fear of violence, abandonment and stigmatization which all involve a male partner. Male involvement facilitates both ART initiation and adherence [11, 12], it increases the probability of mothers delivering at health facility and it enables for a good choice of breastfeeding plan [14–16]. This study aimed in determining magnitude, predictors as perceived by pregnant mothers and effects of male partner involvement in PMTCT services.
|
study
| 99.8 |
This was hospital based cross sectional study involving HIV positive pregnant women in Mwanza urban conducted from October 2013 through January 2014. Mwanza urban has a population of 706,453 people where 237,478 are women in reproductive age group of 15 to 49 years [17, 18]. PMTCT services in the region started in 2004 at Nyamagana hospital and currently there are 45 health facilities which provide PMTCT services in the region. Ten out of 45 Health facilities in Mwanza urban were selected by simple random sampling using lottery method. Proportional to size sampling technique was used to obtain the number of study participants from each participating health facility (The size of each facility was the number of PMTCT attendees as per quarter of July-September 2013). We calculated the sample size based on the estimation of male involvement (defined as men who tested for HIV with their spouse at ANC) in the population of 21% , an absolute precision of 6%, design effect of 1.5 and a 5% level of significance. We adjusted the sample size by considering 10% of anticipated non response. Therefore the total sample size was estimated at 300 HIV positive pregnant women. All pregnant mothers with at least 18 years who tested for HIV and agreed to be enrolled into PMTCT program were included. A pregnant mother in first or second trimester or if her partner is not alive was excluded from the study. Eligible clients from each Health facility who attended on days of interview were enrolled sequentially into the study. A face to face interview was conducted using a pre tested, semi structured questionnaire to collect information on socio demographic information both for participants and their male partners, partner testing and providing support to the mother and Factors affecting male participation in various PMTCT services as perceived by the mothers. Information on mother disclosure of HIV test result to her partner and any negative consequences after disclosure was also obtained.
|
study
| 100.0 |
Data were entered, cleaned, analyzed using Epi info version 3.5.4. Male partner involvement was a composite measure that included partner testing for HIV, partner support and partner aware of Mother's PMTCT status. Partner aware of mother's HIV status (disclosure) was further evaluated for existence of any form of Gender Based Violence. All independent variables including partner's knowledge were factors perceived by the mother as the respondent. Marital status such cohabiting and married were codes as living together while single and divorce were coded as not living together. Further coding for marital status was married for married couple while cohabiting, divorce and single were coded as not married. Univariate and Bivariate were done. Odds Ratio (OR) was used to quantify the likelihood of Male partner involvement into PMTCT and statistical significance was assessed using P- value and 95% confidence intervals for the odds ratios (OR). All independent variables with P-value <0.2 in the Bivariate analysis were considered for multivariate analysis through logistic regression to assess the effect of each covariate in the final model. Step down procedure for logistic regression was used to identify variables associated with male partner involvement in the final model. Ethical clearance was obtained from the Research Ethics Committee of Muhimbili University of Health and Allied Sciences. At the local level permission was also sought from Mwanza region authority and the respective health facilities authority. A written consent was obtained from each participant before interview.
|
study
| 100.0 |
A total of 300 HIV positive pregnant mothers were interviewed in this study. The mean age of the respondents was 27.5 years with standard deviation of 5.57 years. Majority, 211 (70.3%) of these mothers were married. Median gravidity was three with Inter-quartile range (IQR) of two and four while the median number of living children was two with IQR of one and three. The illiterate rate among study participants was more than 10% (Table 1).
|
study
| 100.0 |
The level of male partner involvement in PMTCT was assesed using a series of questions as indicated in Table 2. Out of 300 participants, only 112 (37%) mothers had shared their HIV status with their partners. Partner`s HIV testing level was 114 (38%) of mothers reported their partners to have tested. A total of 96 (32%) mothers reported to have ever received support from their partners during the index pregnancy. The overall male partner involvement in PMTCT services (include men who scored yes in all the three questions) was 24.7% (74).
|
study
| 100.0 |
Of the 112(37%) mothers who reported to have shared their HIV serostatus with their male partners 18 (16.1%) reported different types of gender abuse. A substantial proportion (39%) of women who experienced gender based violence reported that their partners decided to stay away from them (separation) and 11% stated that they were beaten before their partners went to live away. Beating alone was the second most common form of abuse (Figure 1).
|
study
| 99.94 |
Bivariate analyses were done in order to determine whether perceived factors by mother such as TV/Radio announcement, distance from home to ANC, ANC friendly services, socio cultural norms, mother being proactive, partner knowledge on PMTCT, willingness of a mother to be escorted to ANC and busy partner are associated with male partner involvement in Prevention of Mother to child transmission of HIV infection. Socio-demographic factors of both mother and partner such as marital status, partner being in same religion, polygamist partner and partner's level of education were also assessed. In the bivariate analysis, mothers who reported that their partners have been exposed to TV/Radio announcement addressing PMTCT had three times higher odds of their partners being involved in PMTCT than those who reported no such exposure (COR 2.9 CI 1.4-6.6). Likewise mothers who reported that their partner had knowledge on PMTCT had 20 times higher odds of their partners being involved in PMTCT than those who reported no such knowledge (COR 20.5 CI 5.8-72.8). Mothers who were asking for escort to ANC from their partners were having 38 times more chances for their partners to be involvement in PMTCT services than those were not asking for escort (COR 37.9 CI 11.0-130.6). In additional mothers who live together with their male partners are three times more likely to have their male partners involved in PMTCT services than those who don't live together (COR 3.2 CI 1.5-7.0) while mothers who are married have two fold more likely to have their partners involved in PMTCT services than those who are not married (COR 2.3 CI 1.2-4.6). Also mothers who reported to be in same religion with their partners had six times increased chances of male partner being involved in PMTCT services than those who were not in the same religion (COR 5.7 CI 1.7-19.0). On contrary, mothers who did not want to be escorted to ANC by their partners had 92% less likely to have their male partners involved in PMTCT services (COR 0.08 CI 0.01-0.6). Additionally mothers who reported their male partners to be busy with other daily activities especially on days of ANC had 62% less likely their male partners to be involved in PMTCT services (COR 0.38 CI 0.2-0.7). Furthermore socio-cultural norms and multiple marriages were limiting factors for male involvement in PMTCT [(COR 0.3 CI 0.1-0.9) and (COR 0.44 CI 0.2-0.9)]. Partners who had secondary education and above were 32% less likely to be involved in PMTCT services than those who had primary education and below however this association was not significant (Table 3). On logistic regression, proactive mothers who always ask for escort from their partners ranked the first as a promoting factor for male involvement in PMTCT services. Data show that mothers who always ask for escort from their partners had 28.6 times increased odds of their male partners being involved in PMTCT (AOR 28.6 CI 7.1-116). The second promoting factor for male involvement in PMTCT was perceived partner's knowledge on PMTCT. Data show that mothers who reported that their male partners being knowledgeable on PMTCT had 24.6 increased odds of their male partners being involvement in PMTCT than those mothers whose partners had no such knowledge (AOR 24.6 CI 5.9-102). Mothers whose partners had exposure to TV/Radio announcement addressing PMTCT had 4.6 times increased odds of their male partners being involved in PMTCT than those with no such exposure (AOR 4.6 CI 1.5-14.2) while married status of mother had 3.7 times more likely to have their male partners involved in PMTC (AOR 3.7 CI 1.5-9.4). On the other hand, Mothers who did not want to be escorted by their male partners were 93% less likely to have their male partners involved in PMTCT services than those who wanted escort (AOR 0.07 CI 0.01-0.7) while perceived busy partners were 54% less likely to be involved in PMTCT (AOR 0.46 CI 0.2-0.9). The rest factors which were significant on Bivariate analysis were not significant on logistic regression model (Table 3).
|
study
| 99.94 |
In this study we aimed also at determining the effect of male partner involvement on reported gender based violence. There is a great association of reported gender based violence and male partner involvement as data show that those mothers whose partners are involved into PMTCT are 98% less likely to be involved in violence than those whose partners are not involved into PMTCT (Crude OR 0.02 P < 0.001) (Table 4).
|
study
| 100.0 |
This study was able to determine magnitude of male partner involvement into PMTCT services as well as identifying factors affecting male partner involvement into PMTCT services as perceived by mothers. Furthermore it has demonstrated that male partner involvement has a significant influence on preventing Gender based violence. The magnitude of male partner involvement shown in this study was 24.7%. This findings correlates with that reported by Byamugisha et all in Uganda where 26% of men whose wives were attending ANC at Mbale Regional Referral hospital reported to have full male involvement . Likewise, the magnitude of male involvement in this study is more or less the same as that reported in the bottleneck analysis report of Tanzania e-MTCT of 2012 which showed only 21% male partner involvement , this slight difference might be attributed to under reporting of the routine data used to compile the bottleneck report. However due to differences in definition of male involvement and setting of the study, the magnitude in this study is much higher from 12.5% which was reported by Msuya SE et al in Moshi urban . Studies done in Cameroon, South Africa and Malawi have reported male involvement of less than 20% [23–25]. In the same way as found by Kilewo C. et al in 2001 among women participating in a perinatal trial in Dar es salaam, less than 20% of them had shared their HIV results with their partners eighteen months after diagnosis . This correlates with the findings of this study where the proportion of HIV positive pregnant mothers who have shared their serostatus to their partners was 37%. This could be contributed by low (38%) HIV testing level of male partners as noted also by Falnes EF et al 2010 in northern Tanzania . Tadesse E et al 2004 in Blantyre Malawi found also that women have great trust to their male partners if and only if they are tested for HIV because majority of them choose their partner as their primary confidants after they tested for HIV .
|
study
| 99.94 |
In this study, it has been shown that women have a great role to play in making their male partners involved into PMTCT. Data show that proactive mothers who always ask their partners for escort to ANC are mostly associated with positive impact on male partner involvement. The studies done by Fisaha H et al 2012 in Makelle, Nothern Ethiopia on male partner involvement in PMTCT also found that maternal willingness to inform their husband about availability of testing services at ANC was an independent predictor for male partner involvement . As expected, male partners who are knowledgeable on PMTCT are more likely to adopt PMTCT services. Data in this study reveals that male partners who are knowledgeable on PMTCT are 24 fold more likely to be involved into PMTCT than those who have no knowledge about PMTCT. Many studies have found that lacking PMTCT knowledge among male partners is a major hindrance to their male involvement [29, 30]. Furthermore, this study shows that TV/Radio broadcasts play a major role in encouraging men for PMTCT involvement. This is likely the main source of educating and promoting knowledge of PMTCT in the society. On the other hand factors such as socio cultural norms, polygamist partner, and perceived ANC distance which have been shown by other studies as determinants of male involvement in PMTCT [23, 31, 32] were not found to be significant in this study. This is likely to be due to changes in taboos favored by technological advancement in information sharing. Many people in urban area own TV, Radio and phones which have access to various communication media such as facebook, whats up, twitter to mention a few. People can acquire knew knowledge easily and fast, and this facilitates changes in taboos. Factors like friendly ANC services and partner fearing of HIV test result were inconclusively significant because of having empty numbers in some of the shells. This could require more exploration with large sample size. Male partner involvement into PMTCT services in its real definition as defined by a researcher has a negative impact on Gender Based Violence. In this study, male involvement was associated with reduced odds of GBV by 98%. Likewise, a study done by Semrau K et al in 2005 reported that couple counselling makes a man more responsible for the health of the partner and the family resulting to less blame and discrimination . This indicates that counselling and HIV status disclosure resulting from male involvement reduce the probability for a male to engage in violence.
|
study
| 99.94 |
Despite that to a large extent this study has been able to answer intended research questions also it has some limitations. First in this study male information was extracted using female partner. A use of this approach in one way or another might affect the result in this study because female partner is more likely to have recall bias however the findings of this study correlate with other studies which interviewed men [20, 30]. Second the possibility of desirability bias is also expected because mothers attending PMTCT services are trained on PMTCT hence they tend to answer what they think is expected from them. Thirdly, the cross section nature of this study will not prove if the associated factors of male involvement are actually causal. However, most factors identified are also supported by robust studies done elsewhere. Fourth, we anticipated that the use of pre-prepare structure questionnaire may results into information bias, to minimizes this all interviewers were oriented to the meaning and intention of every question before beginning the study. Finally this study was done into a single selected region in Tanzania this might limit generalizability of its findings.
|
study
| 100.0 |
This study was set out to demonstrate the magnitude of male partner involvement and measure the strength of association with the determinants. Male partner involvement into PMTCT services in Mwanza urban is low. Proactive mothers on asking escort from their partners is the mostly associated promoting factor of male involvement followed by partners' knowledge on PMTCT then hearing TV/Radio announcements addressing PMTCT. Unwillingness of a mother to be escorted by partner is the major hindrance of male involvement into PMTCT services. Male partner involvement is likely to reduce events of gender based violence. TV/Radio announcements addressing PMTCT need to be advocated so as to increase exposure to every man. Likewise women have a role to play in making their men participate in PMTCT activities.
|
study
| 99.94 |
Success in preventing MTCT will not only depend on use of antiretroviral prophylaxis, but also on continuing support to the nursing mothers from their male partners. It has been shown that male partner involvement reduces the risk of vertical transmission and infant mortality by 40% compared to non-involvement .
|
other
| 99.9 |
This study demonstrates the magnitude of male partner involvement and its determinants in PMTCT. Proactive mothers on asking escort from their partners is the mostly associated promoting factor of male involvement followed by partners' knowledge on PMTCT then hearing TV/Radio announcements addressing PMTCT;
|
study
| 99.94 |
Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide that, following its release from axon terminals at the median eminence, stimulates the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the pituitary gland. GnRH can also reach the central nervous system (CNS), as GnRH neurones in the hypothalamus can have axons that extend into other regions of the CNS including the limbic system (Silverman et al., 1987). In addition, GnRH can cross the blood-brain barrier, from the median eminence, into the third ventricle cerebrospinal fluid, albeit with low efficiency (Caraty and Skinner, 2008). GnRH receptor expression has been demonstrated at sites within the CNS (Jennes et al., 1997, Albertson et al., 2009, Schang et al., 2011) and a range of peripheral tissues (Hapgood et al., 2005, Skinner et al., 2009). Thus, when GnRH analogs are used therapeutically in human and veterinary medicine, it is also important to consider the effects at these non-reproductive sites.
|
review
| 99.75 |
As GnRH agonists (GnRHa) result in continued receptor stimulation, as opposed to ultradian cyclic changes, theiradministration initially results in an increase in LH and FSH secretion (‘flare-effect’), followed by the down-regulation of GnRH receptor expression in the pituitary gland and suppression of reproductive axis function (Garner, 1994, Chen and Eugster, 2015). GnRHa is typically prescribed when the suppression of the reproductive axis is required, such as steroid-sensitive conditions like prostate cancer, uterine fibroids and endometriosis (Garner, 1994). In children and adolescents, GnRHa can be prescribed for treatment of central precocious puberty (CPP) (Chen and Eugster, 2015) and gender dysphoria (GD) (Hembree et al., 2009) to temporarily halt reproductive development.
|
review
| 99.9 |
Carel et al. (2009) emphasized the need for investigation of the potential psychological effects associated with peripubertal GnRHa-treatment in CPP. Similarly, the potential effects of GnRHa-treatment on cognition during this important developmental period are not well characterized. Wojniusz et al. (2016) recently demonstrated that peripubertal GnRHa increases emotional reactivity (i.e. emotional and behavioral responses to a fearful situation) in girls with CPP, whereas resting heart rate decreased and this effect was more pronounced with longer durations of GnRHa-treatment. Studies, using an ovine model, have also demonstrated that peripubertal GnRHa-treated rams display increased risk-taking behavior (Wojniusz et al., 2011), altered emotional reactivity (Evans et al., 2012) and reduced long-term spatial reference memory (Hough et al., 2016). Physiological changes within the limbic system have also been reported in this ovine model, as peripubertal GnRHa-treatment alters amygdala volume (Nuruddin et al., 2013a) and the expression of hippocampal genes that are involved in endocrine signaling and synaptic plasticity (Nuruddin et al., 2013b). With this growing body of evidence that peripubertal GnRHa-treatment may affect development of cognitive function, there is now a requirement to investigate whether these effects are reversible when GnRHa-treatment is discontinued.
|
review
| 99.4 |
In the current study, we investigated whether effects from peripubertal GnRHa-treatment, persisted in rams following the discontinuation of treatment. Specifically, we investigated whether the previously reported reduction in long-term spatial memory persists, or if effects on spatial orientation and learning emerge later in life, following the discontinuation of peripubertal GnRHa-treatment.
|
study
| 100.0 |
All animal procedures were conducted at the University of Glasgow Cochno Farm and Research Centre (55° 55′N) under Home Office Regulations (Project License: 60/4422). The experimental animals used in this study were Scottish Mule, Texel cross males, born from same sex litters between 23 March and 12 April 2013. At birth, lambs were assigned to one of the treatment groups described below. Lambs from twin and triplet pregnancies were allocated to different groups, so that only 1 sibling was represented in each treatment group. Puberty was delayed in the GnRHa-Recovery (GnRHa-Rec, n = 25) lambs by subcutaneous implantation of the GnRHa, goserelin acetate (Zoladex 3.6 mg, kindly donated by Astra Zeneca, Macclesfield, UK), every four weeks from 8 to 44 weeks of age (average age of pubertal onset in male sheep is 10 weeks of age (Wood and Foster, 1998) and are expected to be sexually competent within the first year of life). Control (n = 30) and GnRHa-Rec rams were grazed on pasture, except during behavioral trials, when they were housed indoors with ad libitum access to hay or silage, with supplements as deemed necessary by standard management practices. All animals were euthanized at the end of the study period, when they were approximately 2 years of age.
|
study
| 100.0 |
Approximately every four weeks, morphometric data were collected from all animals, including testes size, to monitor the effectiveness of GnRHa to suppress the reproductive axis. Scrotal length and circumference were measured with a tailor tape measure while sheep were held in a sitting-position. Testes size was calculated from the scrotal length × circumference and normalized to body weight. Testes size data at 28 (first breeding season), 44 (first anestrus), 81 (second breeding season) and 99 (second anestrus) weeks of age are presented, as these measurements were nearest to the dates of spatial maze performance assessment.
|
study
| 100.0 |
This study used modifications of the assessment techniques and spatial mazes described previously (Hough et al., 2016), as it follows on from this study on the effects of chronic peripubertal GnRHa-treatment (with/without testosterone supplementation) on spatial orientation, learning and memory in rams from 8 to 45 weeks of age. The current study reports analyses of spatial maze performance data following the discontinuation of GnRHa-treatment at 44 weeks of age. Specifically, performance was evaluated at 83 and 95 weeks of age, and compared to that observed at 41 weeks of age (Hough et al., 2016) when the GnRH-Recovery group was still being treated with GnRHa.
|
study
| 100.0 |
Sheep were individually assessed in spatial maze Layout 1 of Fig. 1 at 41 weeks of age, and in Layout 2 of Fig. 1 at 83 and 95 weeks of age. A change in maze layout was necessary, as some of the sheep were already familiar with the former layout (used in long-term spatial memory assessment at 45 weeks of age as reported by Hough et al., 2016). Each sheep was given three maze attempts within the same day (each attempt separated by ∼2 h) to traverse the maze and reunite with flock members in the audience pen. Approximately 30 sheep were assessed per day and kept in the audience pen throughout the day with ad libitum access to water and hay. During each maze attempt, a sheep was calmly ushered from the audience pen to the start of the maze. Sheep that failed to complete the maze within a 5 min time limit, were ushered back to the audience pen via the maze entrance so that the correct route remained unknown. On the last attempt of the day, unsuccessful sheep proceeded to the audience pen via the quickest route Spatial orientation was assessed as the performance of sheep in the first spatial maze attempt of the day, at each age. Spatial learning was assessed as the performance of sheep over three maze attempts within the same day, at each age.
|
study
| 100.0 |
Spatial performance was individually assessed by recording traverse times (min: s: ms), i.e. the time taken to move from the entrance to the finish line (5 min = incomplete), as well as recoding the progress through the maze as the time difference to move between lines A to E, judged on the placement of a front leg across the line. Emotional reactivity was not assessed in this study, as only a few vocalizations, escape attempts, urinations and defecations were observed at 83 and 95 weeks of age.
|
study
| 100.0 |
Education and confirmation runs were performed, over two days, at 95 weeks of age, in the maze layout used in the assessment of spatial orientation and learning (Layout 2, Fig. 5). The education runs were completed when a sheep was able to traverse the maze within 1 min on two successive attempts. The retention of this ability was tested in the confirmation run, which consisted of two attempts to complete the maze within 1 min. If unsuccessful, sheep repeated the cycle of education and confirmation runs, with a maximum of 3 cycles within the same day. The total number of attempts during the education and confirmation runs was recorded for each ram, together with the quickest traverse time, to serve as a measure of the ease of training.
|
study
| 100.0 |
Retention of long-term spatial memory was assessed 4 weeks after training was completed (99 weeks of age), in the same maze design. Each sheep was given one maze attempt, and traverse times (incomplete = 5 min) and progress through maze zones was recorded. The proportion of time spent in each zone was calculated for each animal as a percentage of total time spent in the maze.
|
study
| 99.94 |
Immediately after assessment of long-term memory, each sheep was given one attempt to traverse a new spatial maze layout (Layout 3, Fig. 1), which contained the same ‘traps’ but in a different order or orientation. Maze traverse times (incomplete = 5 min) and progress through maze zones were recorded.
|
study
| 99.75 |
Data were excluded from analysis where performance was judged to have been compromised because of temporary incapacity, i.e. health concerns. In addition, data were excluded from analysis where animals escaped from the maze area or jumped over internal maze walls. Exclusion of data was done by specifying a missing value for the relevant response variable(s) in that particular maze attempt (number of observations is specified in Fig. 3). All statistical analyses were performed with R software (Version 3.2.1, © 2015 The R Foundation for Statistical Computing Platform) using the RStudio interface (Version 0.99.467, © 2009–2015 RStudio Inc.). Response variables were analyzed using the generalized linear model (glm) function; ram identity was included as an explanatory variable, to account for individual variation across time or respective maze attempts.
|
study
| 100.0 |
The effect of GnRHa on body weight and normalized testes size, were analyzed using a: (1) Two-way ANOVA (Treatment × Age) in year 1 and 2, (2) Two-way ANOVA during breeding and non-breeding season. Effects of age and treatment on spatial orientation were assessed with data from the first attempt of the maze, across all ages (41, 83 and 95 weeks of age), using a two-way ANOVA (Treatment × Age). Effects of treatment on spatial learning, over three consecutive maze attempts, were assessed with two-way ANOVA (Treatment × Maze attempt) at each respective age. One-way ANOVA was used to assess the effects of treatment on the ease of maze training (number of training attempts at 95 weeks of age), as well as traverse times upon completion of training. Effects of treatment on long-term spatial memory were tested by comparison of traverse times at: (1) 99 weeks of age only (one-way ANOVA); (2) 95 (last training attempt) versus 99 (the assessment attempt) weeks of age (two-way ANOVA: Treatment × Time); and 99 (GnRHa-Recovery) versus 45 (during GnRHa-treatment) weeks of age (two-way ANOVA: Treatment × Time). The effect of maze design familiarity was examined by comparison of the traverse time of maze layouts 1 and 2 at 83 weeks of age, or layouts 2 and 3 at 95 weeks of age (two-way ANOVA: Treatment × Maze Layout). The effects of treatment and age on testes size was evaluated with a two-way ANOVA. All statistical tests were followed by a Tukey Honest Significant Difference post hoc test, to assess where significant differences existed between treatment groups. All graphs represent means and standard errors of the mean. Statistical P-values ≤ 0.05 were considered significant.
|
study
| 100.0 |
There was no difference (P > 0.05) in body weight between the Control and GnRHa-treated rams at any age. Peripubertal GnRHa-treatment significantly (P < 0.01) suppressed normalized testes size, compared to Control rams, at both 28 and 41 weeks of age (Fig. 2). In the GnRH-Rec group, normalized testes size increased significantly (P < 0.001) by 25% over the 37 weeks following discontinuation of GnRHa-treatment. Normalized testes size during the breeding season in the second year of life, being 47% higher compared to the breeding season of the previous year. At 81 and 99 weeks of age, there was no difference (P > 0.05) in normalized testes size between Control and GnRHa-Rec groups.
|
study
| 100.0 |
The average time taken for sheep to traverse the maze (Fig. 3) was significantly (P < 0.001) different over the three ages tested. Compared to 41 weeks of age (Layout 1), average traverse times at 83 and 95 weeks of age were 84 and 62% slower, respectively. A different maze layout was used at 83 weeks of age (Layout 2) to that used at 41 weeks of age, and comparison of these two ages only, indicated a tendency (P = 0.082) for average traverse times to be longer at 83 weeks of age; an effect that was seen equally in both groups (Controls: 2.92 ± 0.29 vs. 3.47 ± 0.30 min; GnRHa-Rec: 2.96 ± 0.38 vs. 3.49 ± 0.26 min).
|
study
| 100.0 |
There were no significant effects of treatment, or interaction between the effects of age and treatment, on the proportion of time spent in any of the maze zones (Fig. 4, Attempt 1). Regardless of age (P > 0.05), rams spent the greatest proportion of time in zones C and E. However, rams spent significantly (P < 0.001) more time in zones A and B, and significantly (P < 0.01) less time in zone D, at 95 compared to 83 weeks of age.
|
study
| 100.0 |
The mean times taken to complete the maze across all three attempts within the same day, at each age, are shown in Fig. 3 and the P-value summary is reported in Table 1. There was no significant (P > 0.05) difference in spatial learning between the Control and GnRHa-Rec groups, at any age and no significant (P > 0.05) interaction between treatment and age. Significant improvement in traverse times over the three attempts were seen at 41 (P < 0.01), 83 (P < 0.001) and 95 (P = 0.050) weeks of age, but at 83 weeks of age the improvement (P < 0.001) was greater in the new maze layout with a 29.7% decrease in traverse times from the first to second attempt, and 10.7% from the second to third attempt.
|
study
| 100.0 |
The mean proportion of time spent in each maze zone, for the two groups of animals, are shown in Fig. 4 with the associated P-value summary in Table 1. At 41 weeks of age, there was no effect of treatment on the time spent in any maze zone. At 83 weeks of age, on the first attempt, animals spent most time in zones B, C and E. On the second attempt, the proportion of time spent in each zone was more equal, with the exception of zone E, where animals spent the greatest proportion of time. By the third attempt, the time spent in each zone was approximately equal. These changes in the pattern of maze progression were reflected by a significant (P < 0.01) increase in the proportion of time spent in zones A (1st trap) and D (4th trap), and a significant (P < 0.001) decrease in the proportion of time spent in zone C (3rd trap) over the course of the three attempts. The GnRHa-Rec group spent significantly (P < 0.05) less time in zone B than the Control group, particularly in attempt 2. The GnRHa-Rec group also tended (P = 0.080) to spend less time in zone E in attempt 1, but more time in this zone in attempt 3, compared to Controls.
|
study
| 100.0 |
At 95 weeks of age, progress through the maze during the first attempt followed a similar pattern to that seen at 83 weeks of age, with the most time being spent in zones C and E. On the second attempt, there was a reduction in the proportionate time spent in these zones, together with a statistically significant increase (P < 0.05) in the proportion of time spent in zones A and D, but this pattern change was not as marked as seen at 83 weeks of age. The proportion of time spent in zone C tended (P = 0.057) to decrease with each maze attempt in the Controls, so that they spent nearly equal proportions of time across all zones by attempt 3, but the GnRHa-Rec group still spent most of their time in zones C and E, even in attempt 3.
|
study
| 100.0 |
Fig. 5A depicts the mean traverse times at the end of training (‘Trained < 1min’), and 4 weeks later when performance was assessed in the same maze (‘Long-term memory’) and in a novel maze design (‘Novel maze’). There were no effects of treatment on the number of attempts required to complete the training (Controls 6.6 ± 0.8; GnRHa-Rec 7.1 ± 0.9 attempts) or traverse times at the end of training. Compared to traverse times at the end of training, all animals took significantly (P < 0.001) longer to traverse the maze during long-term memory assessment, and GnRHa-treatment significantly exaggerated this effect (Treatment P = 0.041, Treatment × Time P = 0.041), as GnRH-Rec and Control animals were 2-fold and 1.3-fold slower, respectively.
|
study
| 100.0 |
When animals were tested in a novel spatial maze, the traverse times were similar (P = 0.564) for GnRH-Rec and Control animals. Comparison of individuals’ performances in the long-term spatial memory and novel maze assessments, indicated that all animals took significantly (Familiarity P < 0.001) longer to complete the novel maze, irrespective of treatment group (Treatment × Familiarity P = 0.146). The increase in traverse time was significantly affected by treatment (P = 0.008), whereby Control animals took 1.6-fold longer, and GnRH-Rec animals only took 1.1-fold longer, to complete the novel maze compared to the long-term memory assessment.
|
study
| 100.0 |
The mean proportion of time spent in each maze zone during the long-term spatial memory and novel maze assessments are shown in Fig. 5B and C. During the long-term memory assessment, the proportion of time spent in the maze zones was not different between the GnRHa-Rec and Control groups, although there was a trend (P = 0.079) for the Control group to spend a proportionally greater time in zone B compared to the GnRHa-Rec group.
|
study
| 100.0 |
When animals were tested in the novel maze, progress through the maze zones was not affected by treatment, but animals spent significantly (P < 0.05) less time in the last three zones of the novel maze compared to the ‘familiar’ maze layout used in the long-term spatial memory assessment.
|
study
| 100.0 |
Following the discontinuation of GnRHa-treatment at 44 weeks of age, the reproductive axis was no longer suppressed, as testes size increased to a similar size as untreated rams by 83 weeks of age and was larger at the time of the breeding versus non-breeding season. This provides evidence that endogenous GnRH and gonadal steroid signaling was restored. This delayed exposure to gonadal steroid signaling did not alter the speed at which animals completed the spatial tasks during assessments of spatial orientation (i.e. first maze attempt) and learning (progress over 3 same-day maze attempts) after peripubertal GnRHa-treatment had ceased, but affected the manner in how quickly rams moved beyond a specific point within the maze, over three same-day attempts. On assessment of long-term spatial memory at 99 weeks of age, GnRHa-Rec rams took longer to traverse a familiar spatial task, 4 weeks after training, than age-matched Controls, whereas their performance during an unfamiliar spatial task was the same as the Controls. This reduction in long-term spatial memory in GnRHa-Rec animals was also observed prior to the withdrawal of GnRHa-treatment (Hough et al., 2016) and the current study therefore reports that this reduction persisted into adulthood and was not reversed after the discontinuation of peripubertal GnRHa-treatment. A detailed discussion on these main observations follows below.
|
review
| 97.0 |
Although peripubertal GnRHa-treatment did not have profound effects on spatial orientation (i.e. first maze attempt) and learning (i.e. change over 3 same-day maze attempts) during the first year of life (Wojniusz et al., 2011, Hough et al., 2016), peripubertal GnRHa-treatment was associated with alterations to the manner in which rams moved within the maze. This was evidenced by a decreased motivation to complete the maze and increased emotional reactivity within the maze, but did not have a significant effect on the pattern of progression through the maze zones. Supplementation of peripubertal GnRHa-treatment with exogenous gonadal steroids counteracted the effects of GnRHa on motivation and emotional reactivity.
|
study
| 100.0 |
Given the observations in the first year of life, it is not surprising that after discontinuation of GnRHa-treatment in the second year of life, traverse times during assessments of spatial orientation and learning were unaffected in the GnRHa-Rec group. Interestingly, maze progression over three same-day attempts in the GnRHa-Rec group was quicker at 83, but slower at 95 weeks of age, compared to Controls. As breeding and non-breeding seasons are represented at 83 and 95 weeks of age, respectively, it could be indicative that, in the GnRHa-Rec rams, high gonadal steroid levels had a greater impact to improve the ability of rams to learn how to progress beyond a specific point in the spatial maze, compared to the Controls. The manifestation of these differences in maze progression patterns during the second year of life, suggests that the programming of motivational behavior and/or emotional reactivity might be dependent on exposure to gonadal steroids during a critical window of development which coincides with the peripubertal period.
|
study
| 100.0 |
As reported previously (Hough et al., 2016), during the first year of life, peripubertal GnRHa-treated rams required 1.3-fold more training attempts to learn how to complete the spatial maze than untreated rams. This effect was counteracted with testosterone supplementation, indicating that the ease of training was influenced by testosterone, rather than GnRH signaling.
|
study
| 100.0 |
Long-term spatial memory performance was also reduced in peripubertal GnRHa-treated rams, compared to Control rams (1.5-fold). As supplementation of the GnRHa-treatment with exogenous testosterone did not counteract this reduction, it indicated that long-term spatial memory is affected by the loss of GnRH, rather than testosterone, signaling.
|
study
| 100.0 |
Lastly, the assessment of rams in a spatial maze with a novel layout demonstrated that testosterone counteracted the effects of peripubertal GnRHa-treatment to reduce the spatial performance of rams. This indicated that testosterone, rather than GnRH, signaling influenced the ability of rams to solve a spatial task where familiar cues were presented in a novel sequence.
|
study
| 100.0 |
The present study reports that, after GnRHa withdrawal the ease with which animals could be trained to complete a spatial maze in the second year of life was not different from Controls. Thus the deficit in training ability observed during peripubertal GnRHa-treatment was not permanent and a delayed exposure to gonadal steroids was sufficient to restore the speed of spatial maze training.
|
study
| 100.0 |
During the assessment of long-term spatial memory at 99 weeks of age, GnRHa-Rec rams were found to be 1.5-fold slower than Controls in traversing the spatial maze. Interestingly this difference relative to the Controls is of the same magnitude as those reported when animals were receiving GnRHa-treatment (Hough et al., 2016). The maintenance of this difference in spatial performance, following the discontinuation of peripubertal GnRHa-treatment, demonstrated that the effects of peripubertal GnRHa on long-term spatial memory were not reversed and persisted into adulthood, despite the restoration of normal GnRH and gonadal steroid signaling. This result indicates that long-term spatial memory is dependent on changes that occurred during a critical window of development that is sensitive to alterations in GnRH signaling. These changes might be related to neural plasticity and endocrine signaling as suggested by Nuruddin et al. (2013b). Spatial memory can be subdivided into long-term spatial reference memory, which categorizes spatial information according to cues that remain the same between spatial tasks (Olton and Papas, 1979) and working spatial memory, which categorizes information based on the sequence of spatial cues (Olton and Papas, 1979). As the performance of the Control and GnRHa-Rec rams were comparable at 99 weeks of age in the novel maze, which effectively tests spatial working memory, it can be concluded that the observed permanent effects of peripubertal GnRHa-treatment on long-term spatial memory is specific to spatial reference memory. It is also reasonable to argue that the increase in circulating testosterone, which should have accompanied gonadal development in the GnRHa-Rec group, must have been sufficient to eliminate any effects of the peripubertal GnRHa-treatment on spatial working memory.
|
study
| 99.94 |
To our knowledge, this is the first report that the effects of peripubertal GnRHa-treatment to reduce long-term spatial reference memory will persists following GnRHa withdrawal and suggests that these effects are permanent. Studies on the use of GnRHa in adult humans (Freedland et al., 2009, Green et al., 2000) have reported impaired cognition and memory loss, which were ascribed to the associated loss of estrogen and/or testosterone. Beer et al. (2006) reported that long-term memory was reduced, in terms of compromised immediate and delayed verbal memory, in men with prostate cancer and androgen deprivation (primarily GnRHa-mediated) compared to untreated healthy Controls. Another study, on elderly men with prostate cancer and GnRHa-mediated androgen deprivation (Salminen et al., 2004), reported that a decline in testosterone was associated with slower visuo-motor speed, slower reaction times relating to working memory, reduced recognition speed and delayed recall of letters, but improved object recall. These observations support the conclusion of the current ovine study that spatial working memory is reduced by the loss of testosterone, and that restoration of testosterone, either via replacement therapy (Hough et al., 2016) or gonadal development following GnRHa-withdrawal, results in normal function. However, deficits in long-term spatial reference memory did not improve with exposure to endogenous testosterone and are likely permanently altered by the changes in GnRH signaling that occurred during the peripubertal period. In so doing, it identifies the peripubertal period as a critical window of development with regard to spatial memory, in which GnRH signaling is involved. The observation that peripubertal GnRHa-treatment is associated with permanent changes in brain development raises particular concerns about the cognitive changes associated with the prolonged use of GnRHa-treatment in children and adolescents.
|
study
| 99.8 |
Limited studies have looked at the long-term effects of GnRHa-treatment on cognition in children and adolescents. One study reported that 3-year GnRHa-treatment of girls with early pubertal onset was associated with a 7% reduction in IQ (Mul et al., 2001). This effect was hypothesized to be attributed to the suppression of sex steroids and their effect on brain development which resulted in a more age-appropriate IQ (Mul et al., 2001). A recent study of girls treated with GnRHa for CPP, reported that they exhibited increased emotional reactivity and a decreased resting heart rate (Wojniusz et al., 2016). Interestingly, the changes in heart rate observed in that study were dependent upon the duration of GnRHa-treatment. Taken with the results of this study – in which it is indicated that peripubertal GnRHa-treatment affects aspects of cognitive function – it may be worth considering the duration of the GnRHa-treatment in children and adolescents, and limiting it where possible (Hayes, 2016).
|
review
| 99.4 |
The results of this study when considered with those of Hough et al., 2016 suggest that there is a critical period of brain development associated with the peripubertal period. For early onset GD, GnRHa is prescribed from childhood, throughout the adolescent period and into early adulthood, when an informed decision can be made about gender reassignment. This prolonged treatment that may encompass such critical developmental periods, however, may be justified by the increased risk of life-threatening behaviors (e.g. risk taking and suicide attempts), the effects of which could outweigh any minor cognitive and psychological impacts of GnRHa-treatment (Grossman and D’Augelli, 2007, Vance et al., 2014). For CPP, GnRHa-treatment commences from the time of diagnosis of early pubertal onset (8 or 9 years of age for girls or boys, respectively) and continues until approximately 11 years of age (Carel et al., 2009), with the main goal to increase predicted adult height by allowing more time for growth prior to the fusion of bones during puberty. However, this goal has been primarily achieved when GnRHa-treatment commenced with pubertal onset < 6 years of age (Carel et al., 2009, Hayes, 2016). The question then remains whether GnRHa exposure can be limited in children with pubertal onset between 6 to 8 or 9 years of age, or if GnRHa-treatment can be discontinued earlier than 11 years of age.
|
review
| 78.9 |
While continuous GnRHa-treatment may limit psychological problems in CPP, the decision to commence GnRHa-treatment needs further exploration (Carel et al., 2009) as proper parental and physician support might be an alternative intervention (Hayes, 2016). It has been suggested by Steinberg (2004) that a special window of susceptibility to psychosocial and psychopathological conditions exists during adolescence, which is created by the temporal gap between the onset of puberty and late adolescence. At the onset of puberty there are changes in the limbic system and increasing reward sensitivity, whereas the maturation of the prefrontal cortex is slower and age-dependent so that self-regulatory systems are only developed in late adolescence. In this regard, early-maturing children would experience a greater vulnerability towards risk-taking and novelty-seeking behavior, as well as psychopathological conditions, than children that enter puberty at an older age (Steinberg, 2004). This is evidenced by correlations between early pubertal onset and a relatively young age of first sexual intercourse, increased incidence of drug and alcohol abuse, as well as increased risk of sexual abuse (Mul and Hughes, 2008, Kim and Lee, 2012, Hayes, 2016). Against this background, the reduction in long-term spatial memory following peripubertal GnRHa, as observed in the rams in this study, might not outweigh the risks involved in withholding this treatment, but provides evidence that some cognitive functions may be irreversibly altered by peripubertal GnRHa-treatment. Further investigation on other potential cognitive and psychosocial effects are required – including whether these effects are reversible or sexually dimorphic – to assist in making informed decisions about the timing of the commencement and discontinuation of peripubertal GnRHa-treatment.
|
study
| 99.9 |
Spatial orientation and learning performance (i.e. traverse times) were not different from untreated rams, when assessed at 83 and 95 weeks of age, following the discontinuation of peripubertal GnRHa-treatment at 44 weeks of age. However, the effects of peripubertal GnRHa-treatment to increase emotional reactivity, persisted into the second year of life after GnRHa-treatment had been discontinued, because the manner in which rams moved through the maze (i.e. maze progress pattern) over multiple same-day maze attempts differed from the Controls. Interestingly, these aspects of how the rams progressed through a maze appeared to be dependent on the level of gonadal steroid exposure (i.e. quicker maze progression during breeding season vs. slower maze progression during non-breeding season).
|
study
| 100.0 |
The reduction in long-term spatial memory induced by peripubertal GnRHa-treatment persisted in rams into adulthood even after GnRHa-treatment was discontinued. Development of this cognitive function is, therefore, likely to occur during a critical window of development, which may reflect a time-limited period of hippocampal plasticity. Perturbations in GnRH signaling during this peripubertal period may also have long lasting effects on other brain areas and/or aspects of cognitive function.
|
study
| 100.0 |
Laboratory medicine, including diagnostic laboratory microbiology, pathology, hematology, and other testing, has been increasingly included in medical school pre-clinical curricula . This addition has been made necessary by growing expenditures due to inappropriate or excessive diagnostic test ordering by physicians [1, 2]. Despite the inclusion of laboratory medicine to curricula in 52% of the United States allopathic medical schools , it has recently been reported that graduating medical students are not prepared to properly order and interpret laboratory diagnostic tests upon advancement in their medical training [4, 5]. To combat the lack of ability to properly interpret laboratory tests, various sample curricula have been proposed. These suggestions include early exposure to laboratory medicine, the use of small group discussions, and hands-on workshops [6, 7]. However, data demonstrating which educational method yields the greatest student comprehension is lacking .
|
review
| 99.75 |
This issue is common to most course subjects taught at medical schools throughout the United States, yet what makes laboratory medicine unique amongst the pre-clinical sciences is that this field has both cognitive and psychomotor components. The cognitive aspect is knowledge based, and it tracks advancement from simply remembering terms towards the ultimate goal of mastery . The psychomotor component is hands-on, and it is used to develop a learner’s ability to become competent with specific skills . Laboratory medicine fits within both of these domains, as the interpretation of diagnostic laboratory tests is cognitive, while the performance of the test is psychomotor. Because of this duality, the outcomes of training laboratory medicine can be explored in interesting ways to determine whether teaching the psychomotor component can actually aid the cognitive aspects.
|
other
| 99.9 |
The Gram stain is a simple yet commonly used laboratory technique that differentially classifies bacteria by the structure of its cell wall into two large groups: Gram positive and negative. This technique is a useful model to teach laboratory medicine, since it is a standard bacteriology method that pre-clinical students will learn and sometimes perform during their clinical rotations. Hence, we designed a randomly controlled study to determine whether hands-on Gram stain experience increases a student’s ability to interpret the results of this procedure. When comparing a cohort of students taking a hands-on laboratory workshop, a typical pre-clinical lecture, or a small focus group discussion, we hypothesized that the group of students who were immersed in a hands-on setting will achieve higher competence after taking an assessment quiz on the Gram stain procedure compared to the other educational methods. This proof of principle study suggests that practical training of laboratory medical techniques assists in the basic understanding of diagnostic testing by pre-clinical students and may facilitate the integration of the cognitive and psychomotor components for their learning.
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study
| 99.94 |
Study volunteers were first year medical students at NYIT College of Osteopathic Medicine (NYITCOM). Thirty participants were initially enrolled in the study, with ten assigned to each of three separate groups. A total of four participants in both the workshop (n = 1) and discussion (n = 3) groups withdrew at various points in the study, resulting in a total of 26 subjects. Subjects were randomly assigned into three groups: a workshop group (average age: 23.9; 3 males and 6 females), a small discussion group (average age: 23.2; 1 male and 6 females), and a lecture group (average age: 24.4; 2 males and 8 females) (Fig 1). To minimize randomization bias, subjects who reported extensive microbiology education or professional experience were excluded from the study. Effective randomization minimized the likelihood of one group having a greater competency baseline than the other groups. All study participants attended or streamed a lecture entitled “Bacteria: Structure and Physiology” where they learned about the structure of bacterial cell walls and the basic principles of the Gram stain procedure. This lecture was taught in 50 minutes and is a component of the course “Foundations of Osteopathic Medicine” given to the first year NYITCOM medical students as part of their curriculum. In addition to the lecture, the workshop group participants also completed a 1 hour practical exercise where they performed the Gram stain procedure and observed their stained slides using an upright Olympus CH2 light microscope (Olympus, Tokyo, Japan) under the guidance of a microbiology professor. The discussion group met for 1 hour with a microbiology instructor, interpreted pre-stained slides observed under a microscope and discussed why the bacteria appeared as they did, as well as attending the lecture. Both the workshop and discussion groups were free to ask the facilitator questions about the technique protocol, interpretation of the results, and their significance. The lecture group did not have an intervention, and their lecture-only learning followed the same Gram stain education method of the typical NYITCOM student. All participants were given the opportunity to watch a 10 minutes online NYITCOM-produced instructional video that guided viewers through each step of the Gram stain protocol. Additionally, they were instructed for 30 minutes in biosafety practices (BSL-1 and 2) for teaching laboratories as described by the American Society for Microbiology (ASM) published guidelines (http://www.asm.org/index.php/education2/22-education/8308-new-version-available-for-comment-guidelines-for-best-biosafety-practices-in-teaching-laboratories). Each participant had previously taken an undergraduate microbiology course and been trained in the manipulation of BSL-1 microbes. For this study, BSL-2 guidelines were followed by the participants in the laboratory including standard laboratory practices (e.g. hand washing before and after the activity), personal protection requirements (e.g. safety glasses, gloves, coat, closed-toe shoes), and laboratory physical space requirements (e.g. biological safety cabinet (recommended), sink, eyewash station, lockable door to the room, etc.). The Institutional Review Board at NYITCOM approved this study (Protocol number: BHS 1138). All subjects provided verbal informed consent to participate in this study. Written consent was waived by the IRB because the study represented minimal risk for the participants.
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study
| 99.94 |
The Difco/BBL Gram stain kit (BD, Franklin Lakes, NJ) was used for performing the Gram stain procedure. The kit consists of 4 bottles of 1X solutions of crystal violet, Gram’s iodine, acetone-alcohol and safranin. The microscope slides, for use in this study, were prepared using fresh cultures of Staphylococcus aureus strain 6538 (a Gram-positive coccus; ATCC, Manassas, VA) and Klebsiella pneumoniae strain 13882 (a Gram-negative bacillus; ATCC) that had been maintained and grown for 24 h at 37°C on tryptic soy agar plates (BD). These BSL-2 microbes were selected because they are medically important pathogens and relevant to the participants. Smears of each bacterium were made onto separate slides and these were heat-fixed just prior to their use by the study participants. The kit reagents and the microscope slide preparations were examined before being used in the study to ensure their performance accuracy and to provide the expected staining outcomes. The Gram stain procedure was performed following the protocol described in a standard microbiology laboratory manual .
|
study
| 100.0 |
One week after the study interventions, the participants took an 11-question quiz (S1 Quiz), which assessed their understanding of Gram stain procedure, its significance, and interpretation. Ten questions were multiple-choice with each one worth one point. One closed-ended question listed each step of the Gram stain procedure and asked the participants to place each step in the correct order, and this question was worth 1 point (correct or incorrect) on the quiz. The highest score possible on the quiz was 11 points. Each question was categorized as memorization (Questions 1, 2, and 8), understanding (Questions 3–7), or analysis (Questions 9–11), and mean scores for each group within each category were calculated and analyzed.
|
study
| 100.0 |
Statistical analyses were obtained utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA) software. P values were calculated by analysis of variance (ANOVA) and were adjusted by use of the Bonferroni correction. P values of <0.05 were considered significant.
|
study
| 99.9 |
To assess whether a practical workshop would enhance a cohort of first year medical students’ competency in comprehension and interpretation of Gram stain’s results, subjects were tested using a quiz, and mean score performances were compared to other cohorts of students taking a traditional lecture or small focus group discussion (Fig 2). On average, the workshop cohort demonstrated a significantly higher score (mean: 9.33; range: 7–11) than the discussion (mean: 7.57; range: 6–10) (P < 0.01) and lecture (mean: 7.4; range: 5–9) (P < 0.01) cohorts. There was no difference in score between the discussion and lecture groups. Notably, in the workshop group, there were 5 students with ≥ 10 correct questions whereas only 1 student in the discussion and none in the lecture groups had a similar number of correct questions.
|
study
| 100.0 |
Students randomly assigned to a practical workshop group demonstrated higher average quiz score than those students in a discussion or lecture groups. Each symbol represents a subject, bars signify the average (workshop, n = 9; discussion, n = 7; lecture, n = 10) for each experimental condition, and error bars indicate standard deviations. P value significance (*, P < 0.01; NS, not significant) was calculated by ANOVA and adjusted by use of the Bonferroni correction.
|
study
| 99.94 |
To determine the efficacy of each educational tool (e.g. workshop, discussion, or lecture) used in this study, the questions included in the quiz were categorized within three cognitive areas: memorization, understanding, and analysis. The percentage of correct questions per category for each cohort was calculated and compared to each other (Table 1). Students in the workshop group evinced a higher percentage of correct questions in each category compared to the other groups. In the memorization category, we observed that students in the workshop group (88.89%) had a significantly higher percentage of correct questions relative to students in the lecture group (50%) (P<0.05). Although there was a trend when the percentage of correct questions in the workshop group (88.89%) was compared to the discussion group (66.67%), it was not statistically significant. There was no statistical difference in the understanding and analytical categories when the cohorts were compared. The mean percentages of correct questions in the understanding category for students assigned to the workshop, discussion, and lecture groups were 91.11%, 74.29%, and 86%, respectively. Likewise, the mean percentages of correct questions in the analysis questions were 70.37%, 61.90%, and 53.33% for students assigned to the workshop, discussion, and lecture cohorts, respectively.
|
study
| 100.0 |
A major challenge in medicine is making the leap from the medical training years to patient encounters in the clinical setting. The ability of recently graduated medical doctors to order diagnostic tests and interpret their results has been scrutinized, suggesting that medical schools do not properly prepare students for this critical task. Despite the efforts implemented by medical schools in including laboratory medicine in their curriculum, there is a dearth of information on the teaching methods that yield the best students’ learning outcomes. Therefore and as a proof of principle, we asked whether practical experience performing a laboratory technique such as the Gram stain increases competence in the comprehension and interpretation of the procedure by first year medical students.
|
other
| 99.75 |
Our findings demonstrated that hands-on experience considerably enhances students’ laboratory technique competency in comprehension and memorization compared to those exposed to other learning methods. It is conceivable the workshop cohort’s high scores were heavily influenced by the amount of time students spent in close proximity with an instructor who explained the stain procedure step by step. In this regard, five out of nine students attending the workshop obtained ≥10 correct questions. However, the discussion cohort group spent an equal number of classroom hours with the same instructor and only 1 student had a score of 10. In contrast, the students in the lecture group did not spend additional time with the instructor, and this cohort did not have any students with a score >9. These results reveal that the practical experience of the workshop, rather than the additional exposure to the Gram stain didactic material, was important in scoring high on the assessment quiz compared to the other groups. These outcomes are consistent with other studies that suggest the importance of active learning and student-centered pedagogy, especially in the sciences [12, 13]. While previous education-based research has identified peer discussion as a factor that improves academic competence , it is clear that in our study a practical experience provides a unique and advantageous learning aspect in students’ laboratory technique competency, given that there was no difference in the mean quiz score between the discussion and lecture groups and considering that the participants have comparable educational preparation. Previous studies in pedagogical methods have demonstrated that students exposed to hands-on practical experiences have a deeper understanding of the concepts taught in the classroom than the students who only have lecture-based lessons . Our findings support the idea that hands-on learning is more likely to engage students, but also that it can actually boost comprehension in diagnostic techniques.
|
study
| 99.94 |
To better understand the impact of the different teaching methods utilized in this study, we clustered the questions of the assessment in various cognitive categories including memorization, understanding, and analysis. We found that the workshop cohort of students had better percentages of correct questions in every category than students assigned to the discussion or lecture groups. Particularly, we observed that hands-on experience provided by the workshop significantly enhanced students memorization. Interestingly, students having only a lecture showed a better understanding of the procedure than the discussion group. Furthermore, the majority of the students regardless of their cohort demonstrated lower percentage of correct answers to the analysis questions, suggesting that perhaps more efforts should be allocated by instructors to extensively explain the interpretation of the diagnostic test results to students. Likewise, the fact that only half of the students in the lecture group answered correctly the analysis questions indicates that involvement of students in active learning and practical experiences are necessary for mastering diagnostic techniques. Nonetheless, it is important to describe several limitations in implementing hands-on training sessions into medical school curricula. For example, multiple instructors are usually needed to keep low student to teacher ratio, particularly in medium to large size medical schools to maximize student learning. In this regard, complications such as scheduling difficulties and additional preparation time may arise due to other instructors’ professional responsibilities, whether they are researchers, administrators, or teachers of several courses . This is a major challenge in medical education given that some teachers may opt-out of participating in project-based or hands-on learning because of the extra time and preparation required to set those lessons up.
|
study
| 99.94 |
Despite the Gram stain being an important medical laboratory technique, our study is limited and one cannot extrapolate the results presented here to the broad variety of diagnostic tests used in the clinical setting. Similarly, it may be difficult to extrapolate our findings to a greater population, since the act of volunteering for this study by the participants demonstrates a possible eagerness to learn that other students may not possess. Nevertheless, this report establishes a proof of principle to carry out similar studies in the future in order to understand the importance of practical interventions during medical doctors and other health professionals training, especially in diagnostic testing and analysis. Students’ early exposure to diagnostic techniques preferably as undergraduates or at the initial steps of their medical education may enhance their critical thinking, diagnostic skills, and clinical decision-making.
|
study
| 99.75 |
Extracellular vesicles (EVs) are microparticles produced by all cell types in physiological and pathological conditions. Currently, two main types of EVs have been described, microvesicles (MVs) and exosomes, whose differences reside in the dimension as well as in the mechanisms underlying their generation. MVs are particles of 150–500 nm budding from the plasma membrane, whereas exosomes are nanovesicles of 20–100 nm formed intracellularly. Actually, the molecular mechanisms participating in EV biogenesis are known only in part. As far as their functions are concerned, two main hypotheses have been proposed: (i) EVs may act as a system for cell-to-cell communication by shuttling active biomolecules among cells, (ii) they could serve as vehicles for discharging damaged molecules (lipids and proteins) out of the cell, lightening the ubiquitin-proteasomal system as well as lysosomes in case of overload. In recent years, the interest for EVs increased exponentially starting from the observation that in many different diseases their number is significantly increased in the body fluids of patients (1–5). For this reason, EVs are currently under intense investigation for a possible employment in the clinical practice as prognostic biomarkers. Recently, our group reported MVs, derived from myeloid cells (monocytes, macrophages, and microglia), to be significantly upregulated in the cerebrospinal fluid (CSF) of patients with neuroinflammatory disorders, in particular of those suffering from the most severe forms of multiple sclerosis (6). The stimuli responsible for the production of such MVs from myeloid cells are not known, although the role of extracellular ATP (exATP) and its receptor P2X7 has been widely proven, albeit only in vitro; whether exATP recapitulates this effect also in vivo needs to be defined.
|
study
| 99.94 |
Here, we describe the ability of both pro-and anti-inflammatory cytokines, the most represented class of soluble molecules orchestrating inflammatory processes, to enhance the release of MVs from myeloid cells. Additionally, we found this process to be unrelated to that induced by exATP through the activation of its receptor P2X7, but strictly dependent on transcription. Moreover, by using the mouse model of multiple sclerosis, the experimental autoimmune encephalomyelitis (EAE), we found that injection of imipramine, a well-established inhibitor of MV release mediated by exATP (7), did not affect the number of myeloid MVs in the CSF of such mice, with respect to controls. Overall, these findings might suggest the existence of a pathway stimulated by cytokines, but alternative to that centered on the exATP/P2X7 signaling axis, which could be involved in the release of myeloid MVs during inflammatory conditions.
|
study
| 100.0 |
CHME-5 and BV2 cells were cultured in Dulbecco Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum, penicillin-streptomycin (100 U/ml) and 2 mM l-glutamine. Transfections were carried out by Lipofectamine LTX (Invitrogen) according to the manufacturer’s instructions; the p277.pCCLsin.hPGK plasmid encoding for farnesyl-GFP (f-EGFP) was provided by Prof. Luigi Naldini (Università Vita-Salute San Raffaele, Milano). Peripheral blood mononuclear cells were separated from whole blood by density gradient centrifugation (Ficoll-PaqueTM Plus, GE Healthcare) and CD14+ monocytes were purified by immunomagnetic beads [immunomagnetic MicroBeads (MACS® Miltenyi Biotec)]. Monocytes were stimulated with cytokines 18 h after seeding. Blood samples came from three healthy donors recruited among the lab workers, who signed an appropriate informed consent. The study was approved by the local Ethical Committee.
|
study
| 100.0 |
The following antibodies were used: rabbit anti-P2X7 (Alomone Labs), mouse anti-flotillin-1 (BD Bioscience), and rabbit anti-Ki67 (Novocastra). Phalloidin-488 was used to stain F-actin; the monoclonal antibody against desmoyokin-AHNAK (dA) was a gift of Prof. Jacopo Meldolesi (Università Vita-Salute San Raffaele, Milano, Italy). Rabbit anti-Alix (Millipore), goat anti-CD63 (Biorbyt), mouse anti-COX-IV (Cell Signaling Technology), rabbit anti-cleaved caspase-3 (Cell Signaling Technology), β-actin (Sigma). Oxidized ATP (oxATP), brilliant blue G (BBG), probenecid, imipramine, siramesine and actinomycin D (actD) and ethidium bromide were purchased from Sigma-Aldrich; ARL67156 and SR11302 were from Tocris. 10Panx was from Innovagen and 11R-VIVIT from Calbiochem. WP631 was a gift of Dr. Cinthia Farina (San Raffaele Scientific Institute, Milano, Italy).
|
other
| 99.9 |
Interleukin-4 (IL-4), gamma-interferon (IFN-γ), interleukin-5 (IL-5), interleukin-13 (IL-13), interleukin-23 (IL-23), interleukin-27 (IL-27), transforming growth factor-beta (TGF-β), interleukin-6 (IL-6, R&D), and tumor necrosis factor-α (TNF-α Peprotech) were used at a final concentration of 20 ng ml−1, for 24 h (unless otherwise stated). ATP was used at 1 mM for 5–30 min. In all experiments, pharmacological inhibitors were added to the cell medium, composed by DMEM supplemented with vesicle-depleted serum, 1 h before the addition of cytokines and 24 h before that of ATP, and maintained throughout the treatments: in this way the effects of the inhibitors on cytokine and ATP treatments can be compared.
|
study
| 100.0 |
CHME-5 cells were washed twice with Krebs-Ringer Hepes (KRH) solution (125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM KH2PO4, 25 mM HEPES, 1 mM CaCl2, 6 mM glucose) and maintained for 15 min in the presence of ethidium bromide (15 µM, Sigma-Aldrich). ATP (1 mM) was added and the cells were imaged by using an Axiovert A1 microscope (Karl Zeiss) equipped with an Axio Cam ICm1 and a HPX 120 V lamp. Cells treated with ethidium but not with ATP were used as a control.
|
study
| 99.94 |
Ca2+ influx was assessed by imaging using Fluo-4AM as a Ca2+ indicator; briefly, CHME-5 cells were washed two times with KRH solution, then incubated in the dark with Fluo-4AM (4 µM in KRH) at room temperature for 45 min. Pluronic acid (20% w/v) was used for keeping Fluo-4AM in solution. Afterward, cells were washed two times in KRH and incubated for 20 min at 37°C for allowing the complete de-esterification of the indicator. After an additional wash in KRH, cells were treated or not with ATP (1 mM) and imaged under an Axiovert A1 microscope (Karl Zeiss). Untreated cells were used as a control for determining the baseline fluorescence of Fura-4AM.
|
study
| 99.94 |
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