Depletion of preexisting B-cell lymphoma 2-expressing senescent cells before vaccination impacts antigen-specific antitumor immune responses in old mice

The age-related decline in immunity reduces the effectiveness of vaccines in older adults. Immunosenescence is associated with chronic, low-grade inflammation, and the accumulation of senescent cells. The latter express Bcl-2 family members (providing resistance to cell death) and exhibit a pro-inflammatory, senescence-associated secretory phenotype (SASP). Preexisting senescent cells cause many aging-related disorders and therapeutic means of eliminating these cells have recently gained attention. The potential consequences of senescent cell removal on vaccine efficacy in older individuals are still ignored. We used the Bcl-2 family inhibitor ABT-263 to investigate the effects of pre-vaccination senolysis on immune responses in old mice. Two different ovalbumin (OVA)-containing vaccines (containing a saponin-based or a CpG oligodeoxynucleotide adjuvant) were tested. ABT-263 depleted senescent cells (apoptosis) and ablated the basal and lipopolysaccharide-induced production of SASP-related factors in old mice. Depletion of senescent cells prior to vaccination (prime/boost) had little effect on OVA-specific antibody and T-cell responses (slightly reduced and augmented, respectively). We then used a preclinical melanoma model to test the antitumor potential of senolysis before vaccination (prime with the vaccine and OVA boost by tumor cells). Surprisingly, ABT-263 treatment abrogated the vaccine's ability to protect against B16 melanoma growth in old animals, an effect associated with reduced antigen-specific T-cell responses. Some, but not all, of the effects were age-specific, which suggests that preexisting senescent cells were partly involved. Hence, depletion of senescent cells modifies immune responses to vaccines in some settings and caution should be taken when incorporating senolytics into vaccine-based cancer therapies.     

Short, interspersed, and repetitive DNA sequences in Spiroplasma species

Small fragments of DNA from an 8-kbp plasmid, pRA1, from a plant pathogenic strain of Spiroplasma citri were shown previously to be present in the chromosomal DNA of at least two species of Spiroplasma. We describe here the shot-gun cloning of chromosomal DNA from S. citri Maroc and the identification of two distinct sequences exhibiting homology to pRA1. Further subcloning experiments provided specific molecular probes for the identification of these two sequences in chromosomal DNA from three distinct plant pathogenic species of Spiroplasma. The results of Southern blot hybridization indicated that each of the pRA1-associated sequences is present as multiple copies in short, dispersed, and repetitive sequences in the chromosomes of these three strains. None of the sequences was detectable in chromosomal DNA from an additional nine Spiroplasma strains examined.     

Unmasking Heavily O-Glycosylated Serum Proteins Using Perchloric Acid: Identification of Serum Proteoglycan 4 and Protease C1 Inhibitor as Molecular Indicators for Screening of Breast Cancer

Heavily glycosylated mucin glycopeptides such as CA 27.29 and CA 15–3 are currently being used as biomarkers for detection and monitoring of breast cancer. However, they are not well detected at the early stages of the cancer. In the present study, perchloric acid (PCA) was used to enhance detection of mucin-type O-glycosylated proteins in the serum in an attempt to identify new biomarkers for early stage breast cancer. Sensitivity and specificity of an earlier developed sandwich enzyme-linked lectin assay were significantly improved with the use of serum PCA isolates. When a pilot case-control study was performed using the serum PCA isolates of normal participants (n = 105) and patients with stage 0 (n = 31) and stage I (n = 48) breast cancer, higher levels of total O-glycosylated proteins in sera of both groups of early stage breast cancer patients compared to the normal control women were demonstrated. Further analysis by gel-based proteomics detected significant inverse altered abundance of proteoglycan 4 and plasma protease C1 inhibitor in both the early stages of breast cancer patients compared to the controls. Our data suggests that the ratio of serum proteoglycan 4 to protease C1 inhibitor may be used for screening of early breast cancer although this requires further validation in clinically representative populations.     

Value of human amniotic epithelial cells in tissue engineering for cornea

Human amniotic epithelial cells (hAECs) are potentially one of the key players in tissue engineering due to their easy availability. The aim of the present study was to develop an optimal isolation and transportation technique, as well as to determine the immunophenotype and epithelial gene expression of hAECs. Amnion was mechanically peeled off from the chorion and digested with trypsin-ethylenediaminetetraacetic acid. The isolated hAECs were cultured in medium containing 10 ng/mL epidermal growth factor until P4. The epithelial gene expression, cell surface antigen and protein expression of hAECs were analyzed by quantitative polymerase chain reaction, flow cytometry and immunocytochemistry. hAECs were also cultured in adipogenic, osteogenic and neurogenic induction media. The best cell yield of hAECs was seen in the digestion of 15 pieces of amnion (2 × 2 cm) and isolated 30 min after digestion with trypsin. F12:Dulbecco's modified eagle medium was the best medium for short term storage at 4 °C. hAECs expressed CD9, CD44, CD73 and CD90, and negligibly expressed CD31, CD34, CD45 and CD117. After serial passage, CK3, CK19 and involucrin gene expressions were upregulated, while p63, CK1 and CK14 gene expressions were downregulated. Sustained gene expressions of integrin β1 and CK18 were observed. At initial culture, these cells might have stem-like properties. However, they differentiated after serial passage. Nonetheless, hAECs have epithelial stem cell characteristics and have the potential to differentiate into corneal epithelial cells. Further investigations are still needed to elucidate the mechanism of differentiation involved and to optimize the culture condition for long term in vitro culture.     

Role of Trehalose Synthesis in Ralstonia syzygii subsp. indonesiensis PW1001 in Inducing Hypersensitive Response on Eggplant (Solanum melongena cv. Senryo-nigou)

Ralstonia syzygii subsp. indonesiensis (Rsi, former name: Ralstonia solanacearum phylotype IV) PW1001, a causal agent of potato wilt disease, induces hypersensitive response (HR) on its non-host eggplant (Solanum melongena cv. Senryo-nigou). The disaccharide trehalose is involved in abiotic and biotic stress tolerance in many organisms. We found that trehalose is required for eliciting HR on eggplant by plant pathogen Rsi PW1001. In R. solanacearum, it is known that the OtsA/ OtsB pathway is the dominant trehalose synthesis pathway, and otsA and otsB encode trehalose-6-phosphate (T6P) synthase and T6P phosphatase, respectively. We generated otsA and otsB mutant strains and found that these mutant strains reduced the bacterial trehalose concentration and HR induction on eggplant leaves compared to wild-type. Trehalose functions intracellularly in Rsi PW1001 because addition of exogenous trehalose did not affect the HR level and ion leakage. Requirement of trehalose in HR induction is not common in R. solanacearum species complex because mutation of otsA in Ralstonia pseudosolanacearum (former name: Ralstonia solanacearum phylotype I) RS1002 did not affect HR on the leaves of its non-host tobacco and wild eggplant Solanum torvum. Further, we also found that each otsA and otsB mutant had reduced ability to grow in a medium containing NaCl and sucrose, indicating that trehalose also has an important role in osmotic stress tolerance.     

Brm Inhibits the Proliferative Response of Keratinocytes and Corneal Epithelial Cells to Ultraviolet Radiation-Induced Damage

Ultraviolet radiation (UV) from sunlight is the primary cause of skin and ocular neoplasia. Brahma (BRM) is part of the SWI/SNF chromatin remodeling complex. It provides energy for rearrangement of chromatin structure. Previously we have found that human skin tumours have a hotspot mutation in BRM and that protein levels are substantially reduced. Brm−/− mice have enhanced susceptibility to photocarcinogenesis. In these experiments, Brm−/− mice, with both or a single Trp53 allele were exposed to UV for 2 or 25 weeks. In wild type mice the central cornea and stroma became atrophic with increasing time of exposure while the peripheral regions became hyperplastic, presumably as a reparative process. Brm−/−, Trp53+/−, and particularly the Brm−/− Trp53+/− mice had an exaggerated hyperplastic regeneration response in the corneal epithelium and stroma so that the central epithelial atrophy or stromal loss was reduced. UV induced hyperplasia of the epidermis and corneal epithelium, with an increase in the number of dividing cells as determined by Ki-67 expression. This response was considerably greater in both the Brm−/− Trp53+/+ and Brm−/− Trp53+/− mice indicating that Brm protects from UV-induced enhancement of cell division, even with loss of one Trp53 allele. Cell division was disorganized in Brm−/− mice. Rather than being restricted to the basement membrane region, dividing cells were also present in the suprabasal regions of both tissues. Brm appears to be a tumour suppressor gene that protects from skin and ocular photocarcinogenesis. These studies indicate that Brm protects from UV-induced hyperplastic growth in both cutaneous and corneal keratinocytes, which may contribute to the ability of Brm to protect from photocarcinogenesis.     

High yield lipase-catalyzed synthesis of Engkabang fat esters for the cosmetic industry

Engkabang fat esters were produced via alcoholysis reaction between Engkabang fat and oleyl alcohol, catalyzed by Lipozyme RM IM. The reaction was carried out in a 500 ml Stirred tank reactor using heptane and hexane as solvents. Response surface methodology (RSM) based on a four-factor-five-level Central composite design (CCD) was applied to evaluate the effects of synthesis parameters, namely temperature, substrate molar ratio (oleyl alcohol: Engkabang fat), enzyme amount and impeller speed. The optimum yields of 96.2% and 91.4% were obtained for heptane and hexane at the optimum temperature of 53.9 °C, impeller speeds of 309.5 and 309.0 rpm, enzyme amounts of 4.82 and 5.65 g and substrate molar ratios of 2.94 and 3.39:1, respectively. The actual yields obtained compared well with the predicted values of 100.0% and 91.5%, respectively. Meanwhile, the properties of the esters show that they are suitable to be used as ingredient for cosmetic applications.