Decreased Levels of Circulating CD4+CD25+Foxp3+ Regulatory T Cells in Patients with Primary Antiphospholipid Syndrome

Introduction

CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cell dysfunction has been documented in various autoimmune disorders, but not in antiphospholipid syndrome (APS) so far.

Methods

In this cross-sectional study, we aim to investigate CD4⁺CD25⁺Foxp3⁺ Treg cells, CD3⁺CD19^? T cells and CD3^?CD19⁺ B cells in patients with primary APS and healthy controls. Cell subtypes were immunophenotyped using specific monoclonal antibodies (anti-CD3 CY5, anti-CD4 FITC, anti-CD25, anti-Foxp3, anti-CD19 PE) and flow cytometry.

Results

Twenty patients with APS and 20 age- and sex-matched controls were studied. The percentage of total lymphocytes, activated Th cells (CD4+CD25+), Treg cells and CD3^?CD19⁺ B cells were found significantly lower in APS patients as compared to controls (all p?<?0.05).

Conclusion

A dysfunction in CD4⁺CD25⁺Foxp3⁺ Treg cells may represent one of the mechanisms leading to autoimmunity in APS patients. The decreased number of CD3^?CD19⁺ B cells of APS patients warrants further elucidation.     

8-Isoprostane, prostaglandin E2, C-reactive protein and serum amyloid A as markers of inflammation and oxidative stress in antiphospholipid syndrome: a pilot study

Objective

To test the inflammation and oxidative stress hypothesis in antiphospholipid syndrome (APS) patients and to identify possible associations with clinical and laboratory features of the disease.

Methods

Serum amyloid A (SAA), C-reactive protein (CRP), 8-isoprostane and prostaglandin E2 (PGE) were assayed in the sera of 45 APS patients and then compared to control groups made up of 15 antiphospholipid antibody (aPL) negative patients with systemic lupus erythematosus, 15 aPL negative subjects with pregnancy-related morbidity, 15 aPL negative patients with thrombosis, 15 subjects with persistently positive aPL with no signs or symptoms of APS, and 15 healthy volunteers from among the hospital staff.

Results

APS patients showed significantly higher CRP (p?=?0.01), SAA (p?p?=?0.05) and PGE2 (p?=?0.001) plasma levels as compared to controls. Among APS subjects, significantly higher 8-isoprostane and PGE2 levels were observed in patients with triple positivity for aPL (lupus anticoagulant, anticardiolipin and anti-beta2-glycoprotein I antibodies) compared to APS patients with single or double aPL positivity.

Conclusion

Both inflammation and oxidative stress, as measured by SAA, CRP, 8-isoprostane and PGE2, occur in APS and seem to be related to triple positivity for aPL.     

An optimized high-performance liquid chromatography (HPLC) method for benzoylmesaconine determination in Radix Aconiti Lateralis Preparata (Fuzi, aconite roots) and its products

Background

KHU14, an ethanolic extract of Radix Gentianae Macrophyllae (Qinjiao), Rhizoma Coptidis (Huanglian) and Citri Unshiu Pericarpium (Wenzhou migan) was tested for its anti-inflammatory effects.

Methods

Three out of 20 herbs were found to have anti-inflammatory effects. The formulation of these herbs, i.e. KHU14 was tested for croton oil-induced ear edema, carrageenan-induced paw edema, acetic acid-induced capillary permeability, cotton pellet and delayed type hypersensitivity.

Results

KHU14 exhibited anti-inflammatory effects in animal models of acute and chronic inflammation. The anti-inflammatory activity of KHU14 observed was comparable to that of celecoxib. KHU14 inhibited the production of NO and PGE₂ in LPS/IFN-gamma-stimulated peritoneal macrophages, and reduced edema and the amount of infiltrated cells in animal models.

Conclusion

KHU14 exhibited anti-inflammatory effects as demonstrated in typical immunological tests for anti-inflammation in vitro and in vivo.     

SN50 enhances the effects of LY294002 on cell death induction in gastric cancer cell line SGC7901

Introduction

In the previous study, we found that the inhibition of phosphatidylinositol 3-kinase (PI3K) by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 induced SGC7901 cell death in vitro. We did not know whether SN50, which is a specific inhibitor of nuclear factor κB (NF-κB), could increase the cell death induction of gastric cancer of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 in vitro, and we also wanted to know the mechanism of it, which might be applied to clinical tumor therapy.

Material and methods

The 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cytotoxic effects of the drugs. Mitochondrial membrane potential was measured using the fluorescent probe JC-1. Hoechst 33258 staining was used to detect apoptosis and necrosis morphological changes after {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and/or SN50 treatment. Expression of p53, PUMA and Beclin1 were determined with real-time polymerase chain reaction (RT-PCR) analysis. We used transmission electron microscopy to identify ultrastructural changes in SGC7901 cells after {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and/or SN50 treatment.

Results

In this study, we found that treating the human gastric cancer cells SGC7901 with SN50 could significantly enhance the effects of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 on inducing cell death after 24 h, compared to the control group (p < 0.05). Detection of mitochondrial potential and transmission electron microscopic examination indicated that the rate of cell death increased progressively. The expression of p53, PUMA and Beclin1 was up-regulated.

Conclusions

The NF-κB inhibitor SN50 could enhance the role of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 on inducing cell death of human gastric cancer cells SGC7901, which might be a promising new approach to gastric cancer therapy.     

Implantation of neural stem cells embedded in hyaluronic acid and collagen composite conduit promotes regeneration in a rabbit facial nerve injury model

Background

The mouse is an important and widely utilized animal model for bone marrow transplant (BMT) translational studies. Here, we document the course of an unexpected increase in mortality of congenic mice that underwent BMT.

Methods

Thirty five BMTs were analyzed for survival differences utilizing the Log Rank test. Affected animals were evaluated by physical examination, necropsy, histopathology, serology for antibodies to infectious disease, and bacterial cultures.

Results

Severe bacteremia was identified as the main cause of death. Gastrointestinal (GI) damage was observed in histopathology. The bacteremia was most likely caused by the translocation of bacteria from the GI tract and immunosuppression caused by the myeloablative irradiation. Variability in groups of animals affected was caused by increased levels of gamma and X-ray radiation and the differing sensitivity of the two nearly genetically identical mouse strains used in the studies.

Conclusion

Our retrospective analysis of thirty five murine BMTs performed in three different laboratories, identified C57BL/6NCr (Ly5.1) as being more radiation sensitive than B6.Cg-Ptprc^a/NCr (Ly5.2). This is the first report documenting a measurable difference in radiation sensitivity and its effects between an inbred strain of mice and its congenic counterpart eventually succumbing to sepsis after BMT.     

Helicobacter pylori: a ROS-inducing bacterial species in the stomach

Background

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been reported to impact gastric inflammation and carcinogenesis. However, the precise mechanism by which Helicobacter pylori induces gastric carcinogenesis is presently unclear.

Aim

This review focuses on H. pylori-induced ROS/RNS production in the host stomach, and its relationship with gastric carcinogenesis.

Results

Activated neutrophils are the main source of ROS/RNS production in the H. pylori-infected stomach, but H. pylori itself also produces ROS. In addition, extensive recent studies have revealed that H. pylori-induced ROS production in gastric epithelial cells might affect gastric epithelial cell signal transduction, resulting in gastric carcinogenesis. Excessive ROS/RNS production in the stomach can damage DNA in gastric epithelial cells, implying its involvement in gastric carcinogenesis.

Conclusion

Understanding the molecular mechanism behind H. pylori-induced ROS, and its involvement in gastric carcinogenesis, is important for developing new strategies for gastric cancer chemoprevention.     

Very Early Onset Inflammatory Bowel Disease Associated with Aberrant Trafficking of IL-10R1 and Cure by T Cell Replete Haploidentical Bone Marrow Transplantation

Purpose

Loss-of-function mutations in IL10 and IL10R cause very early onset inflammatory bowel disease (VEO-IBD). Here, we investigated the molecular pathomechanism of a novel intronic IL10RA mutation and describe a new therapeutic approach of T cell replete haploidentical hematopoietic stem cell transplantation (HSCT).

Methods

Clinical data were collected by chart review. Genotypes of IL10 and IL10R genes were determined by Sanger sequencing. Expression and function of mutated IL-10R1 were assessed by quantitative PCR, Western blot analysis, enzyme-linked immunosorbent assays, confocal microscopy, and flow cytometry.

Results

We identified a novel homozygous point mutation in intron 3 of the IL10RA (c.368-10C > G) in three related children with VEO-IBD. Bioinformatical analysis predicted an additional 3′ splice site created by the mutation. Quantitative PCR analysis showed normal mRNA expression of mutated IL10RA. Sequencing of the patient’s cDNA revealed an insertion of the last nine nucleotides of intron 3 as a result of aberrant splicing. Structure-based modeling suggested misfolding of mutated IL-10R1. Western blot analysis demonstrated a different N-linked glycosylation pattern of mutated protein. Immunofluorescence and FACS analysis revealed impaired expression of mutated IL-10R1 at the plasma membrane. In the absence of HLA-identical donors, T cell replete haploidentical HSCT was successfully performed in two patients.

Conclusions

Our findings expand the spectrum of IL10R mutations in VEO-IBD and emphasize the need for genetic diagnosis of mutations in conserved non-coding sequences of candidate genes. Transplantation of haploidentical stem cells represents a curative therapy in IL-10R-deficient patients, but may be complicated by non-engraftment.     

Different inflammatory stimuli in the footpad of mice influence the kinetics of resident peritoneal cells

Objectives

Evidence from the literature that inflammation is a systemic biological phenomenon prompted us to investigate whether inoculation of different irritants to the footpad of mice might influence the kinetics of resident peritoneal cells.

Methods

Mice were inoculated in the footpad at different time intervals with Mycobacterium bovis bacillus Calmette-Guerin (BCG), Ehrlich ascitic tumor cells or lipopolysaccharide (LPS), and resident peritoneal cells were analyzed by flow cytometry.

Results

The results indicate that different stimuli induced different responses in resident peritoneal cells. FoxP3 positive regulatory T cells increased drastically in number after BCG inoculation. Conversely, tumor cell inoculation induced a decrease in FoxP3-positive T cells in the peritoneal cavity, although this effect was not statistically significant. Results also show that cells from the paw migrate to the popliteal lymph node and to the peritoneal cavity. Yet, there are cells in the peritoneal cavity that migrate to the popliteal lymph node.

Conclusion

These data show that cells from the peritoneal cavity are influenced by pathologies in remote regions of the animal. How this novel phenomenon influences overall immune responses, courses of infection and tumor growth are open to further investigation.     

Preventive effects of Flos Perariae (Gehua) water extract and its active ingredient puerarin in rodent alcoholism models

Background

Radix Puerariae is used in Chinese medicine to treat alcohol addiction and intoxication. The present study investigates the effects of Flos puerariae lobatae water extract (FPE) and its active ingredient puerarin on alcoholism using rodent models.

Methods

Alcoholic animals were given FPE or puerarin by oral intubation prior or after alcohol treatment. The loss of righting reflex (LORR) assay was used to evaluate sedative/hypnotic effects. Changes of gama-aminobutyric acid type A receptor (GABA_AR) subunits induced by alcohol treatment in hippocampus were measured with western blot. In alcoholic mice, body weight gain was monitored throughout the experiments. Alcohol dehydrogenase (ADH) levels in liver were measured.

Results

FPE and puerarin pretreatment significantly prolonged the time of LORR induced by diazepam in acute alcoholic rat. Puerarin increased expression of gama-aminobutyric acid type A receptor alpha1 subunit and decreased expression of alpha4 subunit. In chronic alcoholic mice, puerarin pretreatment significantly increased body weight and liver ADH activity in a dose-dependent manner. Puerarin pretreatment, but not post-treatment, can reverse the changes of gama-aminobutyric acid type A receptor subunit expression and increase ADH activity in alcoholism models.

Conclusion

The present study demonstrates that FPE and its active ingredient puerarin have preventive effects on alcoholism related disorders.     

B Cell Immunity in Allergic Nasal Mucosa Induces T helper 2 Cell Differentiation

Background

The pathogenesis of allergic diseases is to be further understood. Recent studies indicate that B cells are involved in the immune regulation. The present study aimed to investigate the role of B cells in the initiation of skewed T helper (Th)2 polarization.

Methods

The surgically removed nasal mucosal specimens from 24 patients with allergic rhinitis (AR) and 22 patients with non-AR (nAR) were collected. B cells isolated from the AR nasal mucosa were characterized. The effect of B cells on inducing naïve CD4+ T cells to differentiate into Th2 cells was evaluated with a cell culture model.

Results

Abundant B cells were detected in the nasal mucosa of patients with AR, which also expressed high levels of T cell immunoglobulin mucin domain (TIM)4 and costimulatory molecules. High levels of Staphylococcal enterotoxin B (SEB) were detected in the AR nasal mucosa. Expression of TIM4 could be induced in naïve B cells in the presence of SEB in culture. TIM4+ B cells could induce naïve CD4+ T cells to differentiate into Th2 cells.

Conclusions

TIM4+ B cells from AR nasal mucosa can induce skewed Th2 polarization. It may be a potential therapeutic target in the treatment of AR.

Capsule summary

B cells plays an important role in the initiation of Th2 polarization

Key Messages

? High frequency of B cells exists in nasal mucosa of allergic rhinitis ? These B cells express high levels of TIM4 ? TIM4+ B cells can initiate the skewed Th2 polarization     

Evaluation of signal transduction pathways after transient cutaneous adenoviral gene delivery

Background

Haplo-identical hematopoietic stem cell (HSC) transplantation is very successful in eradicating haematological tumours, but the long post-transplant T-lymphopenic phase is responsible for high morbidity and mortality rates. Clark et al. have described a skin-explant system capable of producing host-tolerant donor-HSC derived T-cells. Because this T-cell production platform has the potential to replenish the T-cell levels following transplantation, we set out to validate the skin-explant system.

Results

Following the published procedures, while using the same commercial components, it was impossible to reproduce the skin-explant conditions required for HSC differentiation towards mature T-cells. The keratinocyte maturation procedure resulted in fragile cells with minimum expression of delta-like ligand (DLL). In most experiments the generated cells failed to adhere to carriers or were quickly outcompeted by fibroblasts. Consequently it was not possible to reproduce cell-culture conditions required for HSC differentiation into functional T-cells. Using cell-lines over-expressing DLL, we showed that the antibodies used by Clark et al. were unable to detect native DLL, but instead stained 7AAD⁺ cells. Therefore, it is unlikely that the observed T-lineage commitment from HSC is mediated by DLL expressed on keratinocytes. In addition, we did confirm expression of the Notch-ligand Jagged-1 by keratinocytes.

Conclusions

Currently, and unfortunately, it remains difficult to explain the development or growth of T-cells described by Clark et al., but for the fate of patients suffering from lymphopenia it is essential to both reproduce and understand how these co-cultures really "work". Fortunately, alternative procedures to speed-up T-cell reconstitution are being established and validated and may become available for patients in the near future.     

Increase in the Expression of CD4 + CD25+ Lymphocytic T Cells in the Indeterminate Clinical Form of Human Chagas Disease After Stimulation With Recombinant Antigens of Trypanosoma cruzi

Purpose

Regulatory T cells are involved in the clinical course of chronic Chagas disease, possibly because they exercise a control in the patient’s inflammatory response to Trypanosoma cruzi. This study analyzed the levels of CD4?+?CD25+ T cells in chronic Chagas disease patients after in vitro stimulation of the peripheral blood mononuclear cells with CRA (Cytoplasmic Repetitive Antigen) or FRA (Flagellar Repetitive Antigen) T. cruzi antigens.

Methods

Groups of patients with the cardiac form and indeterminate form; and non-infected individuals, were selected. The CD4?+?CD25+ T lymphocyte population, as well as the FoxP3 expression and the IL10 production, were evaluated by flow cytometry after stimulation with CRA or FRA.

Result

The IND group presented higher levels of CD4?+?CD25+ T cells than the CARD group. However, there was no evidence of a relationship between FoxP3 and IL10 with any of the chronic forms.

Conclusions

Our results suggest the possible involvement of CD4?+?CD25+ T cells specific to CRA and FRA in controlling the progression of clinical outcomes. Though, further studies are needed to define which mechanisms activate regulatory T cells and lead to pathology control in chronic human Chagas disease.     

Vespa tropica venom suppresses lipopolysaccharide-mediated secretion of pro-inflammatory cyto-chemokines by abrogating nuclear factor-κ B activation in microglia

Objective and design

The present study was aimed to evaluate the anti-inflammatory potentials of Vespa tropica (VT) venom and its isolated peptides. Effects of whole venom and its two peptides (Vt1512 and Vt1386) on lipopolysaccharide (LPS) challenged BV-2 murine microglial cells was evaluated.

Materials

Mouse microglial cell line, BV-2 and crude venom extract as well as purified peptides from VT along with LPS from Salmonella enterica were used for the studies.

Treatment

BV-2 cells were treated with 500 ng/ml of LPS and different doses of crude wasp venom as well as purified peptides.

Methods

We used immunoblotting, cytokine bead arrays and fluorescence activated cell sorter (FACS) to evaluate the levels of various proteins, cytokines and reactive oxygen species (ROS).

Results

Our studies suggest that treatment with whole venom significantly reduces oxidative stress and LPS-stimulated activation of microglia. Also, purified peptides from crude venom exhibited potential anti-inflammatory properties. Further, whole venom was found to be targeting Akt and p38 MAPK pathways, leading to suppressed NF-κB phosphorylation in LPS challenged BV-2 cells.

Conclusions

VT venom possesses anti-inflammatory properties and can be further explored for their therapeutic potential in treating various inflammatory conditions of the central nervous system (CNS).     

VEGF Induces Neuroglial Differentiation in Bone Marrow-Derived Stem Cells and Promotes Microglia Conversion Following Mobilization with GM-CSF

Purpose

Evaluation of potential tropic effects of vascular endothelial growth factor (VEGF) on the incorporation and differentiation of bone-marrow-derived stem cells (BMSCs) in a murine model of anterior ischemic optic neuropathy (AION).

Methods

In the first approach, small-sized subset of BMCs were isolated from GFP donors mice by counterflow centrifugal elutriation and depleted of hematopoietic lineages (Fr25lin^-). These cells were injected into a peripheral vein (1?×?10⁶ in 0.2?ml) or inoculated intravitreally (2?×?10⁵) to syngeneic mice, with or without intravitreal injection of 5 ??g/2??L VEGF, simultaneously with AION induction. In a second approach, hematopoietic cells were substituted by myelablative transplant of syngeseic GFP + bone marrow cells. After 3?months, progenitors were mobilized with granulocyte-macrophage colony-stimulating factor (GM-CSF) followed by VEGF inoculation into the vitreous body and AION induction . Engraftment and phenotype were examined by immunohistochemistry and FISH at 4 and 24?weeks post-transplantation, and VEGF receptors were determined by real time PCR.

Results

VEGF had no quantitative effect on incorporation of elutriated cells in the injured retina, yet it induced early expression of neuroal markers in cells incorporated in the RGC layer and promoted durable gliosis, most prominent perivascular astrocytes. These effects were mediated by VEGF-R1/Flt-1, which is constitutively expresses in the elutriated fraction of stem cells. Mobilization with GM-CSF limited the differentiation of bone marrow progenitors to microglia, which was also fostered by VEGF.

Conclusions

VEGF signaling mediated by Flt-1 induces early neural and sustained astrocytic differentiation of stem cells elutriated from adult bone-marrow, with significant contribution to stabilization retinal architecture following ischemic injury.     

Antimicrobial activities of the methanol extract and compounds from Artocarpus communis (Moraceae)

Background

Artocarpus communis is used traditionally in Cameroon to treat several ailments, including infectious and associated diseases. This work was therefore designed to investigate the antimicrobial activities of the methanol extract (ACB) and compounds isolated from the bark of this plant, namely peruvianursenyl acetate C (1), α-amyrenol or viminalol (2), artonin E (4) and 2-[(3,5-dihydroxy)-(Z)-4-(3-methylbut-1-enyl)phenyl]benzofuran-6-ol (5).

Methods

The liquid microdilution assay was used in the determination of the minimal inhibitory concentration (MIC) and the minimal microbicidal concentration (MMC), against seven bacterial and one fungal species.

Results

The MIC results indicated that ACB as well as compounds 4 and 5 were able to prevent the growth of all tested microbial species. All other compounds showed selective activities. The lowest MIC value of 64 μg/ml for the crude extract was recorded on Staphylococcus aureus ATCC 25922 and Escherichia coli ATCC 8739. The corresponding value of 32 μg/ml was recorded with compounds 4 and 5 on Pseudomonas aeruginosa PA01 and compound 5 on E. coli ATCC 8739, their inhibition effect on P. aeruginosa PA01 being more than that of chloramphenicol used as reference antibiotic.

Conclusion

The overall results of this study provided supportive data for the use of A. communis as well as some of its constituents for the treatment of infections associated with the studied microorganisms.     

Two circulating neutrophil populations in acute inflammation in mice

Objective and design

Recent studies indicate that neutrophils are heterogeneous and may have an immunosuppressive role in addition to their well-known phagocytic and bactericidal function. This study examined neutrophil subpopulations in the circulation, peritoneum, spleen and bone marrow from mice at various time points after induction of acute inflammation.

Material, treatment and methods

Female C57BL/6 mice were injected intraperitoneally with lipopolysaccharide (LPS). Blood, peritoneal, spleen and bone marrow cells were collected and counted and expression of surface molecules and chemokine receptors analyzed with flow cytometry. Chemokine and cytokine concentrations in serum and peritoneal fluid were determined by ELISA.

Results

Neutrophil numbers in the circulation decreased following administration of LPS but reached similar numbers to those prior to inflammation at 8?h. At that time point, two distinct neutrophil populations were present in the circulation. These two neutrophil populations differed in size, granularity and expression of CD11b and Ly6G. Few neutrophils were recruited into the peritoneum until 24?h after administration of LPS at a time when the neutrophils in the circulation had increased their expression of the chemokine receptor CXCR2.

Conclusions

Induction of acute inflammation leads to the appearance of two circulating neutrophil subpopulations, which may differ in their activation state and function.     

Optical imaging of the peri-tumoral inflammatory response in breast cancer

Background

Liposomal doxorubicin (Doxil) is a cytotoxic chemotherapy drug with a favorable hematologic toxicity profile. Its active drug, doxorubicin, has interesting immunomodulatory properties. Here, the effects of Doxil on surviving tumor cell immunophenotype were investigated.

Methods

Using ID8 murine ovarian cancer cells, the immunomodulatory effects of Doxil were studied by measuring its impact on ovarian cancer cell expression of MHC class-I and Fas, and susceptibility to immune attack in vitro. To evaluate the ability of Doxil to cooperate with cancer immunotherapy, the interaction between Doxil and Interleukin 18 (IL-18), a pleiotropic immunostimulatory cytokine, was investigated in vivo in mice bearing ID8-Vegf tumors.

Results

While Doxil killed ID8 tumor cells in a dose-dependent manner, tumor cells escaping Doxil-induced apoptosis upregulated surface expression of MHC-I and Fas, and were sensitized to CTL killing and Fas-mediated death in vitro. We therefore tested the hypothesis that the combination of immunotherapy with Doxil provides positive interactions. Combination IL-18 and Doxil significantly suppressed tumor growth compared with either monotherapy in vivo and uniquely resulted in complete tumor regression and long term antitumor protection in a significant proportion of mice.

Conclusion

These data demonstrate that Doxil favorably changes the immunophenotype of a large fraction of the tumor that escapes direct killing thus creating an opportunity to expand tumor killing by immunotherapy, which can be capitalized through addition of IL-18 in vivo.