Immunologic Effects of An Oral BRAF Inhibitor in a BRAF Wild-Type Murine Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4935-4935
Author(s):  
Gregory S. Vosganian ◽  
Rinke Bos ◽  
Linda Sherman
Keyword(s):  
Immune Response ◽  
Immune System ◽  
Cytokine Production ◽  
Serum Glucose ◽  
Tnf Alpha ◽  
Splenic Lymphocytes ◽  
Wild Type ◽  
Ifn Gamma ◽  
Glucose Levels ◽  
Significant Difference

Abstract Abstract 4935 Introduction: Metastatic melanoma represents a clinical challenge as aggressive therapy often results in unacceptable toxicity and less intensive therapy results in suboptimal clinical outcomes. PLX-4032 is an orally available small molecule which targets constitutively activated BRAFV600E in melanoma cells. BRAF is an important regulator of cell growth, proliferation and migration. We examined the effect of PLX-4032 on the immune system in two BRAF wild type murine systems: the first to determine the effect of PLX-4032 on cytokine production and the second to determine any functional immune effect of PLX-4032 in an insulinoma model. Methods: Effect of PLX-4032 on cytokine production: B10.D2 BRAF WT mice were injected with CD8 lymphocytes expressing a TCR specific for HA518-526 peptide (clone 1) and CD4 lymphocytes expressing a TCR specific for HA110-119 peptide (SFE) on Day 0 and concurrently simulated with appropriate peptide, incomplete Freund's adjuvant, and poly IC. Mice received PLX-4032 200mg/kg or placebo daily for 5 days. On day 6, splenic lymphocytes were harvested and flow cytometry performed to determine CD4 and CD8 IL2, IFN gamma, and TNF alpha production. Effect of PLX-4032 on functional immune response: B10.D2 BRAF WT rat insulin promoter (RIP)-Tag2-hemagglutinin mice bearing insulinoma were injected with clone 1 and SFE lymphocytes on Day 0 and received concurrent stimulation as above. Mice received PLX-4032 200mg/kg or placebo daily for 30 days and serum glucose levels were monitored weekly. Results: Effect of PLX-4032 on cytokine production: We noted no significant difference in the absolute number of splenic lymphocytes recovered in the two groups. There was no significant difference in the percent of CD8+ clone 1 cells producing TNF alpha, IL2 or IFN gamma. There was similarly no significant difference in the percent of CD4+/SFE lymphocytes recovered between the treatment and control groups, nor was there a significant difference in the percent of cells producing IFN gamma. Effect of PLX-4032 on functional immune response: Treatment with PLX-4032 resulted in no significant inhibition of the immune response of transferred lymphocytes against insulinoma. Mice in both groups developed significantly elevated serum glucose levels at the same time and remained diabetic for a similar duration. Discussion: Our data demonstrate that PLX-4032 does not appear to exert a quantitative or functional effect on the immune system in BRAF WT mice. Melanoma therapies often include medications which augment patient immune function or specifically target immune regulation; these data suggest that PLX-4032 does not inhibit normal immune function and therefore may have a role in combination with immunologically directed therapy, possibly resulting in synergistic anti-tumor effect by targeting multiple pathways involved in tumor survival. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Immunological Enhancement Activity of Propolis Flavonoids LiposomeIn VitroandIn Vivo

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Yang Tao ◽  
Deqing Wang ◽  
Yuanliang Hu ◽  
Yee Huang ◽  
Yun Yu ◽  
...  
Keyword(s):  
Immune Response ◽  
Immune System ◽  
Cytokine Production ◽  
Subcutaneous Administration ◽  
Phagocytic Function ◽  
Splenic Lymphocytes ◽  
Adjuvant Activity ◽  
Humoral Immune

The aim of this study was to investigate and assess the effects of propolis flavonoids liposome imposed on the immune system by comparing it to propolis flavonoids and blank liposome.In vitro, the effects of the above drugs on macrophages were assessed by measuring the phagocytic function and cytokine production.In vivo, the immunological adjuvant activity of propolis flavonoids liposome was compared with those of propolis flavonoids and blank liposome. The results showed thatin vitropropolis flavonoids liposome can significantly enhance the phagocytic function of macrophages and the release of IL-1β, IL-6, and IFN-γ. In addition, subcutaneous administration of propolis flavonoids liposome with ovalbumin to mice could effectively activate the cellular and humoral immune response, including inducing higher level concentrations of IgG, IL-4, and IFN-γin serum and the proliferation rates of splenic lymphocytes. These findings provided valuable information regarding the immune modulatory function of propolis flavonoids liposome and indicated the possibility of use of propolis flavonoids liposome as a potential adjuvant.

scholarly journals A Comparative Study on the Effects of Using Hydroalcoholic Extracts of Linum Usitatissimum and Rosa Damascena on Liver Function in Adult Male Rats

2020 ◽  
Vol 26 (1) ◽  
pp. 54-67
Author(s):  
Mohammad Mehdi Padam ◽  
◽  
Ameneh Khoshvaghti ◽  
Keyword(s):  
Liver Function ◽  
Total Protein ◽  
Linum Usitatissimum ◽  
Colorimetric Method ◽  
Serum Glucose ◽  
Male Rats ◽  
Control Group ◽  
Rosa Damascena ◽  
Glucose Levels ◽  
Significant Difference

Aims: Damage to liver tissue and its dysfunction is very important and if left untreated, it can cause serious problems and even death. In this study, we aimed to investigate the effects of the hydroalcoholic extracts of Linum usitatissimum and Rosa damascena on liver enzymes, total protein, bilirubin, albumin, and serum glucose levels. Materials and Methods: This is a non-randomized clinical trial conducted on 42 male rats divided into 6 groups; control group (group 1) received only sufficient water and food, groups 1 and 2 received 300 and 500 mg/ kgB.W Linum usitatissimum extract, groups 3 and 4 received 500 and 1000 mg/ kgB.W Rosa damascena, and group 6 received 100 mg/ kgB.W Linum usitatissimum plus 250 mg/ kgB.W Rosa damascena extracts intraperitoneally for 28 days. After the last injection, the rats were weighed and their blood samples were collected. The study parameters were measured using a colorimetric method by a spectrophotometer, and then were analyzed using ANOVA and Tukey’s test in SPSS V. 25 at a significance level of P<0.05. Findings: There was no significant difference between alanine aminotransferase, alkaline phosphatase, total and direct bilirubin levels in the control group in comparison with other groups (P>0.05). In the groups received Rosa damascena extract, there was a significant difference between total protein and albumin levels compared to the control group (P<0.05). Moreover, there was a significant difference between serum glucose and aspartate aminotransferase in the control group compared to other groups (P<0.05). Conclusion: Linum usitatissimum and Rosa damascena have no negative effect on the liver function. The probability of diarrhea occurrence and the possible effects on the total protein and serum albumin after using Rosa damascena, and the effects of different doses of Linum usitatissimum on the glucose levels should be taken into account.

scholarly journals Lymphokine overproduction in severe aplastic anemia is not related to blood transfusions

Blood ◽  
1989 ◽  
Vol 74 (8) ◽  
pp. 2713-2717 ◽  
Author(s):  
W Hinterberger ◽  
G Adolf ◽  
P Bettelheim ◽  
K Geissler ◽  
C Huber ◽  
...  
Keyword(s):  
Cyclosporin A ◽  
Aplastic Anemia ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Severe Aplastic Anemia ◽  
Tnf Alpha ◽  
Blood Transfusions ◽  
Peripheral Blood Mononuclear ◽  
Ifn Gamma

Abstract The production of interferons (IFNs), IFN-gamma, tumor necrosis factors (TNFs) and TNF-alpha (TNF-alpha) by peripheral blood mononuclear cells (PBMNCs) of untransfused and transfused, but otherwise untreated patients with severe aplastic anemia (SAA) was determined using bioassays and immunoassays. In untransfused and pretransfused SAA patients, spontaneous and lectin-induced production of these cytokines by PBMNCs was strongly enhanced. Cytokine production in untransfused SAA patients did not differ from that in pretransfused patients. Similar relative frequencies of activated (HLA-DR+) lymphocyte subpopulations present in the PBMNCs demonstrated cytokine overproduction per cells. Cytokine production was studied in three SAA patients before and after blood cell transfusions. Spontaneous and lectin-induced production of these cytokines was abnormally high and unaffected by blood transfusions. In another patient exhibiting abnormal cytokine production, the hematopoietic response to cyclosporin- A in vivo was accompanied by normalization of cytokine production in vitro. We conclude that overproduction of IFN-gamma and TNF-alpha by lectin-stimulated PBMNCs is an intrinsic abnormality of SAA unrelated to blood transfusions. Normalization of production of IFN-gamma and TNF- alpha accompanying a clinical response to cyclosporin-A may cautiously be taken as further evidence suggesting a pathogenetic role of cytokine overproduction in SAA.

scholarly journals Enhancement of immune response against Bordetella spp. by disrupting immunomodulation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Monica C. Gestal ◽  
Laura K. Howard ◽  
Kalyan Dewan ◽  
Hannah M. Johnson ◽  
Mariette Barbier ◽  
...  
Keyword(s):  
Immune Response ◽  
Immune System ◽  
Immune Cells ◽  
Lymphoid Tissue ◽  
Protective Immunity ◽  
Inflammatory Cells ◽  
Host Immune System ◽  
Initial Growth ◽  
Wild Type ◽  
Sterilizing Immunity

AbstractWell-adapted pathogens must evade clearance by the host immune system and the study of how they do this has revealed myriad complex strategies and mechanisms. Classical bordetellae are very closely related subspecies that are known to modulate adaptive immunity in a variety of ways, permitting them to either persist for life or repeatedly infect the same host. Exploring the hypothesis that exposure to immune cells would cause bordetellae to induce expression of important immunomodulatory mechanisms, we identified a putative regulator of an immunomodulatory pathway. The deletion of btrS in B. bronchiseptica did not affect colonization or initial growth in the respiratory tract of mice, its natural host, but did increase activation of the inflammasome pathway, and recruitment of inflammatory cells. The mutant lacking btrS recruited many more B and T cells into the lungs, where they rapidly formed highly organized and distinctive Bronchial Associated Lymphoid Tissue (BALT) not induced by any wild type Bordetella species, and a much more rapid and strong antibody response than observed with any of these species. Immunity induced by the mutant was measurably more robust in all respiratory organs, providing completely sterilizing immunity that protected against challenge infections for many months. Moreover, the mutant induced sterilizing immunity against infection with other classical bordetellae, including B. pertussis and B. parapertussis, something the current vaccines do not provide. These findings reveal profound immunomodulation by bordetellae and demonstrate that by disrupting it much more robust protective immunity can be generated, providing a pathway to greatly improve vaccines and preventive treatments against these important pathogens.

scholarly journals Mice that lack the interferon-gamma receptor have profoundly altered responses to infection with Bacillus Calmette-Guérin and subsequent challenge with lipopolysaccharide.

1993 ◽  
Vol 178 (4) ◽  
pp. 1435-1440 ◽  
Author(s):  
R Kamijo ◽  
J Le ◽  
D Shapiro ◽  
E A Havell ◽  
S Huang ◽  
...  
Keyword(s):  
Interferon Gamma ◽  
Receptor Gene ◽  
Interleukin 1 ◽  
Tnf Alpha ◽  
Bacillus Calmette Guerin ◽  
Wild Type ◽  
Necrosis Factor Alpha ◽  
Ifn Gamma ◽  
Lps Challenge ◽  
Gamma Receptor

Mice with a targeted disruption of the interferon gamma receptor gene (IFN-gamma R0/0 mice) and control wild-type mice were inoculated with the Bacillus Calmette-Guérin (BCG) strain of Mycobacterium bovis. BCG infection was not lethal for wild-type mice whereas all IFN-gamma R0/0 mice died approximately 7-9 wk after inoculation. Histological examination at 2 and 6 wk after BCG inoculation showed that livers of IFN-gamma R0/0 mice had higher numbers of acid-fast bacteria than wild-type mice, especially at 6 wk. In parallel, the livers of IFN-gamma R0/0 mice showed a reduction in the formation of characteristic granulomas at 2 wk after inoculation. Injection of lipopolysaccharide (LPS) 2 wk after BCG inoculation was significantly less lethal for IFN-gamma R0/0 mice than for wild-type mice. Reduced lethality of LPS correlated with a drastically reduced production of tumor necrosis factor alpha (TNF-alpha) in the IFN-gamma R0/0 mice. Interleukin 1 alpha (IL-1 alpha) and IL-6 levels in the serum were also significantly reduced in the IFN-gamma R0/0 mice after BCG infection and LPS challenge. The greatly reduced capacity of BCG-infected IFN-gamma R0/0 mice to produce TNF-alpha may be an important factor in their inability to resist BCG infection. These results show that the presence of a functional IFN-gamma receptor is essential for the recovery of mice from BCG infection, and that IFN-gamma is a key element in the complex process whereby BCG infection leads to the sensitization to endotoxin.

The Maternal Immune Response to in Utero Hematopoietic Stem Cell Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 64-64
Author(s):  
Tippi MacKenzie ◽  
Erin Jarvis ◽  
Amar Nijagal ◽  
Tom Le ◽  
Marta Wegorzewska ◽  
...  
Keyword(s):  
Immune Response ◽  
Stem Cell ◽  
Immune System ◽  
B Cells ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
In Utero ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Adaptive Immune

Abstract Abstract 64 In utero hematopoietic stem cell transplantation (IUHSCTx) is a promising treatment strategy for many congenital hematopoietic disorders such as immunodeficiencies. However, clinical applications have been hampered by lack of engraftment, possibly secondary to a host immune response. This has been a conundrum in the field, since the fetus can also be tolerized to allogeneic cells in some circumstances. We hypothesized that it is the maternal immune response which limits engraftment of in utero transplanted cells. Methods: Fetal BALB/c mice at 14 days' gestation were transplanted with age-matched fetal liver (FL) cells (2.5 × 106 cells/fetus) from allogeneic C57B6 mice and levels of circulating donor cell chimerism were determined serially starting at 4 weeks after in utero transplantation. Rates of engraftment (number of chimeric pups/number of surviving pups) and levels of chimerism (donor CD45 cells/total CD45 cells) were compared to controls in which animals were transplanted with congenic cells (C57B6 (CD45.2) fetal hosts transplanted with C57B6 (CD45.1) FL). In order to determine the role of the maternal adaptive immune system, immunodeficient BALB/c.Rag−/− mothers (deficient in T and B cells) were bred to wild type BALB/c males, such that the fetuses (BALB/c.Rag+/−) would be immunocompetent. These fetuses were transplanted with C57B6 FL and rates of engraftment and levels of chimerism in these transplants were compared to those in wild type allogeneic transplants. In order to determine whether the maternal influence is caused by maternal lymphocytes trafficking into the fetus, C57B6 (CD45.2) females were bred to C57B6 (CD45.1) males, such that the fetal cells (CD45.1+/CD45.2+) could be distinguished from maternal cells (CD45.1−/CD45.2+). Fetal blood and tissues were examined for the presence of maternal cells by flow cytometry at various gestational ages. Results: The rate of engraftment after IUHSCTx in control animals transplanted with congenic cells was 14/16 (88%) and average levels of chimerism were 9.9±8.4%. In contrast, the rate of engraftment in wild-type BALB/c fetuses transplanted with allogeneic B6 cells was 11/25 (44%; p<0.05 compared to congenic), and levels of chimerism were 21±19 (p=NS), confirming there is an adaptive immune response to fetal stem cell transplantation. As expected, chimeric animals were tolerant to the donor strain by mixed lymphocyte reaction while injected, non-chimeric animals were sensitized. However, in the absence of a maternal adaptive immune system, rates of chimerism (in immunocompetent BALB/c.Rag+/− pups) increased to 100% (n=10, p<0.05 compared to wild type allogeneic) and levels of chimerism were significantly higher (44±18, p<0.05). Levels of chimerism in engrafted animals declined over time after allogeneic transplantation but not after congenic transplantation, indicating there is a second, late phase immune response to allogeneic cells. However, chimerism levels did not decline in the BALB/c.Rag+/− recipients, suggesting that the maternal immune system has long-lasting effects on the success of fetal transplantation, perhaps by priming the host immune system. In our analysis of maternal/fetal cellular trafficking, we detected maternal lymphocytes in the blood of midgestation fetuses (14±7% at E12.5–E14.5, n=9) which declined gradually and was undetectable after birth. Lineage analysis demonstrated that 45±15 % of maternal cells are Gr-1+ granulocytes and 21±15% are B cells. Trafficking of maternal cells into the fetus was increased following fetal manipulation (injection of PBS < injection of allogeneic HSC). Conclusions: There is an adaptive immune response which limits early engraftment after in utero transplantation of allogeneic cells and leads to a gradual decline in levels of chimerism in engrafted animals. However, in the selective absence of maternal T and B cells, all fetuses transplanted with allogeneic FL cells show long-term, multilineage engraftment and demonstrate donor-specific tolerance. These results indicate that the maternal immune system plays a significant role in the success of fetal HSC transplantation. Cellular trafficking between the mother and fetus may be a mechanism by which maternal lymphocytes encounter cells transplanted into the fetus. Our findings have clinical implications in that the success of IUHSCTx may be improved by harvesting cells from the mother or HLA-matching cells to the mother. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Glycemic Index of Selected Foods in Jamaica

2019 ◽  
pp. 13-16
Author(s):  
Ryan Francis ◽  
Perceval S Bahado-Singh ◽  
Andrew O Wheatley ◽  
Ann Marie Smith ◽  
Helen N Asemota
Keyword(s):  
Glycemic Index ◽  
Glycemic Load ◽  
Psidium Guajava ◽  
Serum Glucose ◽  
Dietary Advice ◽  
Diabetic Patients ◽  
Citrullus Vulgaris ◽  
Glucose Levels ◽  
Significant Difference ◽  
Low Glycemic Index

Background: Fruits, vegetables and legumes for their complex carbohydrates, dietary fiber and micronutrients, should form an essential part of every diet. In order to give good dietary advice to diabetic patients, it is necessary to know the glycemic index of foods commonly consumed locally. The objective of this study was to determine the Glycemic Index (GI) and Glycemic Load (GL) of commonly available and consumed Guava (Psidium guajava), Watermelon (Citrullus vulgaris), Gungo (Cajanus cajan), Papaya (Carica papaya) and tomato (Solanum lycopersicum) in Jamaica. Methods: Ten (10) healthy Jamaican subjects (5 males, 5 females) with mean age 30 ± 2 years and mean BMI 25 ± 1 kg/m2 were recruited to the study. Using a non-blind, crossover design trial, the subjects consumed 50 (or 25) grams of available carbohydrate portions of glucose (standard food) and test foods after an overnight fast and their serum glucose levels were determined at 0, 15, 30, 45, 60, 90 and 120 minutes after the consumption of each test food. Glucose was tested on three separate occasions, and the test foods once. The GI value was calculated geometrically by expressing the Incremental Area Under the Blood Glucose Curve (IAUC) for the test foods as a percentage of each subject's average IAUC for the standard food. Results: The results indicated that the IAUC for Watermelon (95 ± 11) was significantly higher (p<0.05) than that of Tomato (37 ± 12), and Gungo (58 ± 13). The differences in IAUC of Watermelon (95 ± 11), Guava (83 ± 27) and Papaya (80 ± 7) were not statistically significant. Similarly, there was no significant difference in GI among the samples studied. Conclusion: Tomato, Gungo, Papaya Watermelon and Guava were shown to have low glycemic index and glycemic load values.

scholarly journals Modulating activity of interferon-gamma on endotoxin-induced cytokine production in cancer patients

Blood ◽  
1991 ◽  
Vol 78 (12) ◽  
pp. 3254-3258
Author(s):  
A Mackensen ◽  
C Galanos ◽  
R Engelhardt
Keyword(s):  
Cancer Patients ◽  
Interferon Gamma ◽  
Cytokine Production ◽  
Repeated Administration ◽  
Tnf Alpha ◽  
Necrosis Factor Alpha ◽  
Ifn Gamma ◽  
Lps Challenge ◽  
Monocytes And Macrophages

Intravenous (IV) administration of purified lipopolysaccharide (LPS) from Salmonella abortus equi to cancer patients induces the formation of high amounts of endogenous cytokines such as tumor necrosis factor- alpha (TNF-alpha) and interleukin-6 (IL-6). On repeated administration of LPS at 2-week intervals, a marked downregulation of the cytokine response was observed, especially between the first and the second challenge. This study sought to determine whether it would be possible to prevent this downregulation by pretreating patients with interferon- gamma (IFN-gamma), which is known to enhance cytokine production by monocytes and macrophages in vitro. Ten patients with disseminated cancer received a first injection of 4.0 ng LPS/kg. Thereafter, patients were divided into two groups. One group received two further LPS injections (4.0 ng/kg) at 2-week intervals. The second group was pretreated (-12 hours) with 50 micrograms IFN-gamma subcutaneously (SC) before the second and third LPS challenge. To prevent constitutional side effects such as fever and chills, patients received 1,600 mg ibuprofen orally before LPS injection. The results of the current study demonstrate that apart from TNF-alpha and IL-6, two other cytokines, interleukin-8 (IL-8) and granulocyte colony-stimulating factor (G-CSF) are produced in cancer patients in response to LPS. LPS application at 2-week intervals resulted in a transient attenuation of all cytokines (TNF-alpha, IL-6, IL-8, G-CSF) on the second challenge. In the case of TNF-alpha, IL-6, and G-CSF, pretreatment with IFN-gamma not only prevented the downregulation, but enhanced the production of these cytokines to levels higher than those obtained after the first LPS challenge. In contrast, the downregulation of IL-8 remained unaffected by IFN-gamma pretreatment. Further studies are warranted to determine whether the prevention of cytokine downregulation by IFN-gamma following repeated LPS injections is of clinical relevance in respect to the antitumor activity of LPS.

scholarly journals The Effect of Remote Ischaemic Preconditioning on the Immune Response

2021 ◽  
Author(s):  
◽  
Jennifer Mae Williams-Spence
Keyword(s):  
Immune Response ◽  
Cardiac Surgery ◽  
Immune System ◽  
Inflammatory Response ◽  
Direct Effect ◽  
Peripheral Blood ◽  
Serum Levels ◽  
Ischaemic Preconditioning ◽  
Significant Difference ◽  
Remote Ischaemic Preconditioning

<p>Remote ischaemic preconditioning (RIPC) describes the phenomenon where brief intermittent periods of limb ischaemia are used to protect the heart and other organs from subsequent prolonged ischaemic insults. RIPC has been identified as a promising intervention for use during cardiac surgery and has consistently shown a beneficial effect in animal models; however, the results of early clinical trials have not been as successful. The exact mechanisms involved in mediating RIPC have not yet been characterised and a better understanding of the pathways through which RIPC exerts its protective effects will be essential in order to progress the translation of this intervention into the clinical setting. There is increasing evidence that RIPC modifies the inflammatory response, therefore the central aim of the research presented in this thesis was to investigate how RIPC affects the human immune system.  We performed a double-blind randomised controlled trial of RIPC in 96 high-risk cardiac surgery patients and found no evidence that the intervention reduced myocardial injury or altered peri-operative expression levels of the key inflammatory cytokines, interleukin (IL)-6, IL-8, and IL-10, during simple or more complex procedures. There was a trend towards higher levels of IL-6 and IL-8 in the preconditioned patients; however, confounding variables in the trial design and the heterogeneous patient population limited our ability to interpret the results.  We next conducted a paired-analysis trial with 10 healthy male volunteers to assess the direct effect of preconditioning on the early immune response, away from any form of ischaemic injury or comorbidities. We found that RIPC directly and significantly decreased serum levels of the chemokines MIP-1α and MIP-1β, but did not increase the serum concentrations of a range of key cytokines or alter the cytokine producing potential of peripheral blood leukocytes. These findings strongly suggest that a cytokine is not likely to be the humoral mediator associated with transmitting the RIPC protective signal.  RIPC did not alter the immunophenotype or extravasation of peripheral leukocyte populations, or the proliferative and cytokine responses of peripheral blood mononuclear cells (PBMC) to pharmacological, physiological, and antigen-specific stimuli. However, preconditioning did appear to reduce the ability of monocytes and neutrophils to respond to activation signals, as indicated by lower levels of CD11b expression in stimulated cultures, and a significant increase in the basal production of IL-22 was also detected in PBMC cultured for 6 days following preconditioning. These alterations may reduce neutrophil and monocyte tissue infiltration and limit the inflammatory response during the early window of RIPC-induced protection and enhance tissue and wound repair several days later. A multivariate analysis confirmed that there was a significant difference in the response between the control and RIPC treatments and the main contributing factors were identified as changes in neutrophil and T cell activation, serum levels of MIP-1α and β, and production of IL-10 and IL-22 from PBMC cultured for 6 days.  Overall, our results suggest that RIPC has a subtle but direct effect on the systemic innate immune response during the early window of protection in healthy volunteers, whereas the effects on the adaptive immune system seem to be considerably delayed. The changes detected following RIPC are likely to contribute to protection against ischaemia-reperfusion injury but not solely account for the extent of the beneficial effects of RIPC detected in animals. Our findings reinforce the safety profile of this intervention and have defined a number of immune parameters that are altered by preconditioning for focusing future research.</p>

Proteolytically Inactivatable Factor VIII is Less Immunogenic than Factor VIII in a Murine Hemophilia A Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 27-27
Author(s):  
Shannon Meeks ◽  
Ernest T Parker ◽  
Amy L. Dunn ◽  
John F Healey ◽  
Pete Lollar
Keyword(s):  
Immune Response ◽  
Immune System ◽  
Factor Viii ◽  
Binding Site ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Foreign Protein ◽  
C2 Domain ◽  
Wild Type ◽  
Antibody Titers

Abstract Abstract 27 Patients with hemophilia A have a congenital deficiency of the factor VIII (fVIII) protein due to a mutation in the fVIII gene that frequently leads to absence of detectable expression of fVIII. Accordingly, the therapeutic replacement fVIII protein potentially is recognized as non-self by the immune system. Thirty percent of patients with severe hemophilia A develop detectable inhibitory anti-fVIII antibodies (inhibitors). Additionally, greater than 90 percent of hemophilia A mice treated with human fVIII develop inhibitors using dosing schedule that mimics use in humans. Because fVIII is an immunologically foreign protein, it might be expected that a hemophilia A patient would make a fVIII inhibitor. However, intravenous injection of soluble proteins in either humans or rodents usually results in tolerance rather than a humoral immune response. One major difference between fVIII and other proteins is that it is released from its large carrier protein von Willebrand factor (VWF) and is potentially exposed to the immune system at sites of active hemostasis and inflammation. Heat-inactivated, denatured fVIII, which maintains all T-cell epitopes but lacks several B-cell epitopes, is less immunogenic than native fVIII, suggesting that fVIII-dependent thrombin generation along the intrinsic pathway of blood coagulation may provide co-stimulatory signals necessary for the immune response (Skupsky BS, Zhang A, Scott DW Blood 2008; 112:1220a). We constructed a B domain-deleted human fVIII mutant, designated fVIIIi, which contains alanine substitutions at two critical thrombin cleavage sites, Arg372 and Arg1689, and purified it to homogeneity. FVIIIi does not develop procoagulant activity and is not released from VWF in response to thrombin. Therefore fVIIIi is less likely than wild-type fVIII to be exposed to the immune system at sites of active hemostasis and inflammation. Additionally, VWF binds to the immunodominant fVIII C2 domain and potentially hides part of fVIII from the immune system. FVIIIi was antigenically intact judging from intact binding to a panel of11 mouse anti-fVIII monoclonal antibodies whose epitope specificity was represented by all five domains of BDD fVIII. The immunogenicity of wild-type fVIII and fVIIIi was compared in a murine hemophilia A model in which groups of 25 mice received 8 weekly injections of physiologic doses of fVIII. Plasma was collected weekly for total anti-fVIII antibody titers by ELISA and one week following the last injection for total anti-fVIII antibody titers, inhibitor titers by Bethesda assay and for epitope mapping. Mice treated with fVIIIi had significantly lower levels of inhibitory as well as total anti-fVIII antibodies than mice treated with wild-type fVIII. Domain mapping using single human domain hybrid human/porcine molecules as ELISA antigens revealed that hemophilia A mice broadly recognized all fVIII domains in response to either wild-type or fVIIIi, although fVIIIi produced less anti-light chain antibodies. Mice in both the wild-type fVIII and fVIIIi groups produced antibodies that recognized the phospholipid-binding site of the C2 domain, even though this site overlaps the VWF binding site on fVIII. There was no difference in the isotype spectrum of the antibodies made to fVIII or fVIIIi. This study indicates that inactivatable fVIII is less immunogenic than native fVIII and suggests that the immunogenicity of fVIII is related either to its interaction with VWF or to events triggered by activation of the coagulation mechanism. Disclosures: No relevant conflicts of interest to declare.

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