CD19 and BAFF‐R can signal to promote B‐cell survival in the absence of Syk

The development and function of B lymphocytes is regulated by numerous signaling pathways, some emanating from the B‐cell antigen receptor (BCR). The spleen tyrosine kinase (Syk) plays a central role in the activation of the BCR, but less is known about its contribution to the survival and maintenance of mature B cells. We generated mice with an inducible and B‐cell‐specific deletion of the Syk gene and found that a considerable fraction of mature Syk‐negative B cells can survive in the periphery for an extended time. Syk‐negative B cells are defective in BCR, RP105 and CD38 signaling but still respond to an IL‐4, anti‐CD40, CpG or LPS stimulus. Our in vivo experiments show that Syk‐deficient B cells require BAFF receptor and CD19/PI3K signaling for their long‐term survival. These studies also shed a new light on the signals regulating the maintenance of the normal mature murine B‐cell pool.     

The diagnostic potential of MPT63‐derived HLA‐A*0201‐restricted CD8+T‐cell epitopes for active pulmonary tuberculosis

MPT63 protein is found only in Mycobacterium tuberculosis complex, including M. tuberculosis and M. bovis. Detection of MPT63‐specific IFN‐γ‐secreting T cells could be useful for the diagnosis of tuberculosis (TB) diseases. In the present study, the HLA‐A*0201 restriction of ten predicted MPT63‐derived CD8 ⁺ T‐cell epitopes was assessed on the basis of T2 cell line and HLA‐A*0201 transgenic mice. The diagnostic potential of immunogenic peptides in active pulmonary TB patients was evaluated using an IFN‐γ enzyme‐linked immunospot assay. It was found that five peptides bound to HLA‐A*0201 with high affinity, whereas the remaining peptides exhibited low affinity for HLA‐A*0201. Five immunogenic peptides (MPT63_18–26, MPT63_29–37, MPT63_20–28, MPT63_5–14 and MPT63_10–19) elicited large numbers of cytotoxic IFN‐γ‐secreting T cells in HLA‐A*0201 transgenic mice. Each of the five immunogenic peptides was recognized by peripheral blood mononuclear cells from 45% to 73% of 40 HLA‐A*0201 positive TB patients. The total diagnostic sensitivity of the five immunogenic peptides was higher than that of a T‐SPOT.TB assay (based on ESAT‐6 and CFP‐10) (93% versus 90%). It is noticeable that the diagnostic sensitivity of the combination of five immunogenic peptides and T‐SPOT.TB assay reached 100%. These MPT63‐derived HLA‐A*0201‐restricted CD8 ⁺ T‐cell epitopes would likely contribute to the immunological diagnosis of M. tuberculosis infection and may provide the components for designing an effective TB vaccine.     

Oxidized LDL induces phosphorylation of non-muscle myosin IIA heavy chain in macrophages

Oxidized LDL (oxLDL) performs critical roles in atherosclerosis by inducing macrophage foam cell formation and promoting inflammation. There have been reports showing that oxLDL modulates macrophage cytoskeletal functions for oxLDL uptake and trapping, however, the precise mechanism has not been clearly elucidated. Our study examined the effect of oxLDL on non-muscle myosin heavy chain IIA (MHC-IIA) in macrophages. We demonstrated that oxLDL induces phosphorylation of MHC-IIA (Ser1917) in peritoneal macrophages from wild-type mice and THP-1, a human monocytic cell line, but not in macrophages deficient for CD36, a scavenger receptor for oxLDL. Protein kinase C (PKC) inhibitor-treated macrophages did not undergo the oxLDL-induced MHC-IIA phosphorylation. Our immunoprecipitation revealed that oxLDL increased physical association between PKC and MHC-IIA, supporting the role of PKC in this process. We conclude that oxLDL via CD36 induces PKC-mediated MHC-IIA (Ser1917) phosphorylation and this may affect oxLDL-induced functions of macrophages involved in atherosclerosis. [BMB Reports 2015; 48(1): 48-53]     

Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model

CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (K_D ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC₅₀ typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.     

Mitochondrial metabolism in cancer stem cells: a therapeutic target for colon cancer

It has been proposed that the selective elimination of cancer stem cells (CSCs) using targeted therapy could greatly reduce tumor growth, recurrence, and metastasis. To develop effective therapeutic targets for CSC elimination, we aimed to define the properties of CSC mitochondria, and identify CSC-mitochondria-specific targets in colon cancer. We found that colon CSCs utilize mitochondrial oxidative phosphorylation (OXPHOS) to produce ATP. We also found that forkhead box protein 1 (FOXM1)-induced peroxiredoxin 3 (PRDX3) maintains the mitochondrial function, and the FOXM1/PRDX3 mitochondrial pathway maintains survival of colon CSCs. Furthermore, FOXM1 induces CD133 (PROM1/prominin 1) expression, which maintains the stemness of colon CSCs. Together, our findings indicate that FOXM1, PRDX3, and CD133 are potential therapeutic targets for the elimination of CSCs in colon cancer. [BMB Reports 2015; 48(10): 539-540]     

CD271 as a marker to identify mesenchymal stem cells from diverse sources before culture

Mesenchymal stem cells, due to their characteristics are ideal candidates for cellular therapy. Currently, in culture these cells are defined by their adherence to plastic, specific surface antigen expression and multipotent differentiation potential. However, the in vivo identification of mesenchymal stem cells, before culture, is not so well established. Pre-culture identification markers would ensure higher purity than that obtained with selection based on adherence to plastic. Up until now, CD271 has been described as the most specific marker for the characterization andpurification of human bone marrow mesenchymal stem cells. This marker has been shown to be specifically expressed by these cells. Thus, CD271 has been proposed as a versatile marker to selectively isolated and expand multipotent mesenchymal stem cells with both immunosuppressive and lymphohematopoietic engraftment-promoting properties. This review focuses on this marker, specifically on identification of mesenchymal stem cells from different tissues. Literature revision suggests that CD271 should not be defined as a universal marker to identify mesenchymal stem cells before culture from different sources. In the case of bone marrow or adipose tissue, CD271 could be considered a quite suitable marker; however this marker seems to be inadequate for the isolation of mesenchymal stem cells from other tissues such as umbilical cord blood or wharton’s jelly among others.     

Evidence for a rapid structural change in TMV-A-protein near neutrality

Titration of TMV-A-protein from pH 8 to 7 (20°C) or raising the temperature from 4° to 20°C (pH 7) produces, within a few minutes, a reversible change in the aromatic region of the CD-spectrum, before any extensive aggregation has taken place. This spectral change is solely a matter of the conditions of the solution and not of the history of the protein. There is no further CD-change during the slow aggregation process. Thus there must be some proton-uptake within the A-protein. The results are discussed with regard to the different interpretations of the role of A-protein or double-disc in the elongation-step of TMV-“in vitro” -self-assembly.     

Effect of palmitoylated prolactin-releasing peptide on food intake and neural activation after different routes of peripheral administration in rats

Obesity is an escalating epidemic, but an effective non-invasive therapy is still scarce. For obesity treatment, anorexigenic neuropeptides are promising tools, but their delivery from the periphery to the brain is complicated by their peptide character. In order to overcome this unfavorable fact, we have applied the lipidization of neuropeptide prolactin-releasing peptide (PrRP), whose strong anorexigenic effect was demonstrated. A palmitoylated analog of human PrRP (h palm-PrRP31) was injected in free-fed Wistar rats by three routes: subcutaneous (s.c.), intraperitoneal (i.p) (both 5 mg/kg) and intravenous (i.v.) (from 0.01 to 0.5 mg/kg). We found a circulating compound in the blood after all three applications with the highest concentration after i.v. administration. This corresponds to the effect on food intake, which was also strongest after i.v. injection. Moreover, this is in agreement with the fact that the expression of c-Fos in specific brain regions involved in food intake regulation was also highest after intravenous application. Pharmacokinetic data are further supported by results obtained from dynamic light scattering and CD spectroscopy. Human palm-PrRP31 analog showed a strong tendency to micellize, and formation of aggregates suggested lower availability after i.p. or s.c. application. We have demonstrated that palm-PrRP influenced food intake even in free fed rats. Not surprisingly, the maximal effect was achieved after the intravenous application even though two orders of magnitude lower dose was used compared to both two other applications. We believe that palm-PrRP could have a potential as an antiobesity drug when its s.c. application would be improved.     

Coincident parasite and CD8 T cell sequestration is required for development of experimental cerebral malaria

Cerebral malaria (CM) is a fatal complication of Plasmodium falciparum infection. Using a well defined murine model, we observed the effect on disease outcome of temporarily reducing parasite burden by anti-malarial drug treatment. The anti-malarial treatment regime chosen decreased parasitaemia but did not cure the mice, allowing recrudescence of parasites. These mice were protected against CM, despite their parasitaemia having increased, following treatment cessation, to levels surpassing that associated with CM in mice not treated with the drug. The protection was associated with reduced levels of cytokines, chemokines, CD8⁺ T cells and parasites in the brain. The results suggest that the development of the immunopathological response that causes CM depends on a continuous stimulus provided by parasitised red blood cells, either circulating or sequestered in small vessels.