Optogenetic engineered umbilical cord MSC-derived exosomes for remodeling of the immune microenvironment in diabetic wounds and the promotion of tissue repair.

BACKGROUND: Angiogenesis and tissue repair in chronic non-healing diabetic wounds remain critical clinical problems. Engineered MSC-derived exosomes have significant potential for the promotion of wound healing. Here, we discuss the effects and mechanisms of eNOS-rich umbilical cord MSC exosomes (UCMSC-exo/eNOS) modified by genetic engineering and optogenetic techniques on diabetic chronic wound repair. METHODS: Umbilical cord mesenchymal stem cells were engineered to express two recombinant proteins. Large amounts of eNOS were loaded into UCMSC-exo using the EXPLOR system under blue light irradiation. The effects of UCMSC-exo/eNOS on the biological functions of fibroblasts and vascular endothelial cells in vitro were evaluated. Full-thickness skin wounds were constructed on the backs of diabetic mice to assess the role of UCMSC-exo/eNOS in vascular neogenesis and the immune microenvironment, and to explore the related molecular mechanisms. RESULTS: eNOS was substantially enriched in UCMSCs-exo by endogenous cellular activities under blue light irradiation. UCMSC-exo/eNOS significantly improved the biological functions of cells after high-glucose treatment and reduced the expression of inflammatory factors and apoptosis induced by oxidative stress. In vivo, UCMSC-exo/eNOS significantly improved the rate of wound closure and enhanced vascular neogenesis and matrix remodeling in diabetic mice. UCMSC-exo/eNOS also improved the inflammatory profile at the wound site and modulated the associated immune microenvironment, thus significantly promoting tissue repair. CONCLUSION: This study provides a novel therapeutic strategy based on engineered stem cell-derived exosomes for the promotion of angiogenesis and tissue repair in chronic diabetic wounds.

A Novel Nonsense Mutation of PHF6 in a Female with Extended Phenotypes of Borjeson-Forssman-Lehmann Syndrome

Borjeson-Forssman-Lehmann syndrome (BFLS) is a rare X-linked disease caused by PHF6 mutations. Classic BFLS has been associated with intellectual disability (ID), developmental delay (DD), obesity, epilepsy, typical facial features and anomalies of fingers and toes. Endocrinological phenotypes and outcome of treatment in this condition remain to be delineated. Here we report a patient who exhibited complete growth hormone deficiency who responded to hormonal treatment but with adverse effects. Horseshoe kidney was present in this patient, which is also atypical in BFLS. A heterozygous nonsense mutation c.673C>T (p.R225X) of PHF6 gene was identified in the patient, inherited from her unaffected mother. Both the patient and her mother showed highly skewed X-inactivation. We reviewed the phenotypes of all reported BFLS cases, and summarized their endocrine presentations. This first report of an Asian patient with BFLS further delineated the genetic and phenotypic spectrum of the syndrome. The adverse effect experienced by the patient suggests caution in the use of growth hormone treatment in this condition.

[Association of Cosmc gene mutation with susceptibility to Henoch-Schönlein purpura in children].

OBJECTIVE: To investigate the presence of Cosmc gene mutation in children with Henoch-Schönlein purpura (HSP) and the association between Cosmc gene mutation and the susceptibility to HSP. MESULTS: Eighty-four children who were diagnosed with HSP between March 2014 and December 2015 were selected as the HSP group. Fifty-eight healthy volunteers matched for age and sex were enrolled as the control group. Fasting venous blood (5 mL) from the two groups was collected in EDTA anticoagulated tubes, followed by the isolation of peripheral blood mononuclear cells (PBMCs) through density gradient centrifugation. Genomic DNA was extracted from PBMCs according to the manufacturer's protocol, and the whole exon region of Cosmc gene was amplified by touch-down polymerase chain reaction (touch-down PCR). The PCR products were identified by 1% agarose gel and sequenced in order to further examine the association between Cosmc gene mutation and the susceptibility to HSP. RESULTS: Sequencing results showed two mutations (c.393T>A and c.72A>G) of Cosmc gene in children with HSP. There were no significant differences in the genotype and allele frequencies at the two loci between the HSP and control groups, and this distribution was not associated with sex. CONCLUSIONS: The mutations c.393T>A and c.72A>G in the exon region of Cosmc gene in children with HSP are not associated with the onset of HSP.

CA215 and GnRH receptor as targets for cancer therapy.

Two monoclonal antibodies (Mabs), RP215 and GHR106, were selected for the preclinical evaluations of anti-cancer drugs targeting various human cancers including those of the ovary, cervix, lung, and liver. Both Mabs were shown to react with pan cancer markers, which are over-expressed on the surface of almost all human cancers. RP215 Mab was shown to react with the carbohydrate-associated epitope(s) of cancer cell-expressed glycoproteins, mainly consisting of immunoglobulin superfamily (IgSF) proteins and mucins, generally known as CA215. GHR106 Mab was generated against the extracellular domain of human GnRH receptor, which is also highly expressed on the cancer cell surface. Preclinical studies were performed to evaluate the efficacy of these two Mabs as anti-cancer drugs for treating human cancers. High tumor specificity of RP215 Mab was demonstrated with immunohistochemical staining studies of various cancer cell lines, as well as normal and cancerous tissue sections. These two Mabs were shown to induce apoptosis as well as complement-dependent cytotoxicity upon treatment to many cultured cancer cells. Significant dose-dependent growth inhibition of tumor cells from several different tissue origins were demonstrated by nude mouse experiments. It was further demonstrated that GHR106 Mab can function as long-acting GnRH analogs in its biological actions. Efforts were made to generate human/mouse chimeric forms of the GHR106 Mab. Based on the results of these preclinical studies, we believe that these two Mabs, in chimeric or humanized forms, can be developed into suitable therapeutic agents for treatment of human cancers as anti-cancer drugs.

Widespread expressions of immunoglobulin superfamily proteins in cancer cells.

RP215 monoclonal antibody (Mab) was shown to recognize a carbohydrate-associated epitope of cancer cell-expressed glycoproteins, known as CA215. Extensive MALDI-TOF MS analysis was performed to search for the molecular identity of CA215. Besides immunoglobulin (Ig) heavy chains, homology to human T-cell receptors (TCR) and Ig-like cell adhesion molecules was also detected. By using RT-PCR and cDNA sequencing, it was observed that as many as 80% of cancer cell lines showed significant levels of gene expressions of TCR-α and TCR-ß. Selected Ig-like cell adhesion molecules such as CD47, CD54, CD58 and CD 147 were also highly expressed among all the cell lines tested. In contrast, co-receptors and co-stimulators of TCR such as CD3, CD4 and CD8 were rarely expressed demonstrating the non-functional nature of TCR in cancer cells. Results of immunohistochemical staining and Western blot assays of cancer cell lines as well as cancerous tissue sections were consistent with these observations. Anti-TCR and anti-human IgG antibodies were shown to induce complement-dependent cytotoxicity and apoptosis of cultured cancer cells indicating the surface nature of Ig-like proteins. Based on these experimental observations, it was hypothesized that the expressions of these immunoglobulin superfamily (IgSF) proteins may be relevant to the immune protection and proliferations of cancer cells during carcinogenesis or cancer progression. Surface-bound TCR-like proteins as well as immunoglobulins may be the potential targets for RP215-based anti-cancer drugs.

Carbohydrate-associated immunodominant epitope(s) of CA215.

RP215 monoclonal antibody (Mab) was initially generated against OC-3-VGH ovarian cancer cells and was shown to react with a cancer-associated carbohydrate epitope in glycoproteins designated as CA215. Additional five high affinity Mabs, designated as RCA-10, -100, -104, -110 and -111, respectively, were generated by using affinity-purified CA215 as the immunogen in this study. All RCA Mabs were found to recognize periodate-sensitive carbohydrate-associated epitope(s) and to pair with RP215 in typical sandwich enzyme immunoassays for the quantification of CA215. When compared with those of RP215, the amino acid sequence homology of the Fab regions ranged from 100% for RCA-100 to 65% for RCA-110, based on which 3 distinct Mab groups were categorized. In vitro TUNEL apoptosis and complement-dependent cytotoxicity assays were performed with these Mabs and found to have comparable inhibitory efficacy to cancer cells. Results of biochemical and immunological assays revealed that RP215, RCA-100 and RCA-10 react with the linear carbohydrate-associated epitope, whereas the others recognize the conformational form of the epitope in CA215. This study has suggested that the unique carbohydrate-associated epitope(s) is immunodominant in mice when immunized with CA215. It remains to be demonstrated if the differential anti-cancer efficacy exists among the distinct groups of these anti-CA215 Mabs.

Identification of cholesterol-regulating genes by targeted RNAi screening.

Elevated plasma cholesterol levels are considered responsible for excess cardiovascular morbidity and mortality. Cholesterol in plasma is tightly controlled by cholesterol within cells. Here, we developed and applied an integrative functional genomics strategy that allows systematic identification of regulators of cellular cholesterol levels. Candidate genes were identified by genome-wide gene-expression profiling of sterol-depleted cells and systematic literature queries. The role of these genes in cholesterol regulation was then tested by targeted siRNA knockdown experiments quantifying cellular cholesterol levels and the efficiency of low-density lipoprotein (LDL) uptake. With this strategy, 20 genes were identified as functional regulators of cellular cholesterol homeostasis. Of these, we describe TMEM97 as SREBP target gene that under sterol-depleted conditions localizes to endo-/lysosomal compartments and binds to LDL cholesterol transport-regulating protein Niemann-Pick C1 (NPC1). Taken together, TMEM97 and other factors described here are promising to yield further insights into how cells control cholesterol levels.

Functions of the siderophore esterases IroD and IroE in iron-salmochelin utilization.

The siderophore salmochelin is produced under iron-poor conditions by Salmonella and many uropathogenic Escherichia coli strains. The production of salmochelin, a C-glucosylated enterobactin, is dependent on the synthesis of enterobactin and the iroBCDEN gene cluster. An E. coli IroD protein with an N-terminal His-tag cleaved cyclic salmochelin S4 to the linear trimer salmochelin S2, the dimer salmochelin S1, and the monomers dihydroxybenzoylserine and C-glucosylated dihydroxybenzoylserine (salmochelin SX, pacifarinic acid). The periplasmic IroE protein was purified as a MalE-IroE fusion protein. This enzyme degraded salmochelin S4 and ferric-salmochelin S4 to salmochelin S2 and ferric-salmochelin S2, respectively. In E. coli, uptake of ferric-salmochelin S4 was dependent on the cleavage by IroE, and independent of the FepBDGC ABC transporter in the cytoplasmic membrane. IroC, which has similarities to ABC-multidrug-resistance proteins, was necessary for the uptake of salmochelin S2 from the periplasm into the cytoplasm. IroE did not function as a classical binding protein since salmochelin S2 was taken up in the absence of a functional IroE protein. IroC mediated the uptake of iron via enterobactin in a fepB mutant. IroE was also necessary in this case for the uptake of ferric-enterobactin, which indicated that only the linear degradation products of enterobactin were taken up via IroC. PfeE, the Pseudomonas aeruginosa IroE homologue, was cloned, and its enzymic activity was shown to be very similar to that of IroE. It is suggested that homologues in other bacteria are also periplasmic IroE-type esterases of siderophores.