Molecular Medicine

Chán ăn tâm thần (AN) là một bệnh lý phức tạp, gây suy nhược, được đặc trưng bởi nỗi sợ hãi mạnh mẽ về việc tăng cân và tập thể dục quá mức. Đây là rối loạn tâm thần gây tử vong cao nhất với tỷ lệ tái phát cao, tuy nhiên, sinh lý bệnh của nó vẫn chưa rõ ràng. Mô hình Chán ăn Dựa trên Hoạt động (ABA) là một mô hình chuột được công nhận rộng rãi về AN, tái hiện tình trạng ăn uống kém và hoạt động quá mức mặc dù trọng lượng cơ thể giảm, nhưng không thể hiện tính mãn tính. Ở đây, chúng tôi đã điều chỉnh mô hình ABA nguyên mẫu nhằm gia tăng thời gian để mất 25% trọng lượng cơ thể cơ bản từ ít hơn 7 ngày lên hơn 2 tuần. Chúng tôi đã sử dụng mô hình này để xác định các gen thay đổi kéo dài sau khi phục hồi cân nặng, đại diện cho một ký ức phiên mã về tình trạng thiếu dinh dưỡng và có thể góp phần vào việc tái phát của AN thông qua phân tích giải trình tự RNA. Chúng tôi tập trung vào mô mỡ vì nó được xác định là vị trí chính của ký ức phiên mã về tình trạng thừa dinh dưỡng. Chúng tôi đã xác định được 300 gen rối loạn điều hòa không hồi phục sau khi phục hồi cân nặng sau ABA, bao gồm Calm2 và Vps13d, những gen này có thể là các yếu tố điều hòa toàn cầu của ký ức phiên mã trong cả tình trạng thừa và thiếu dinh dưỡng mãn tính. Chúng tôi đã chứng minh sự tồn tại của các thay đổi kéo dài trong phiên mã mô mỡ ở chuột ABA sau khi phục hồi cân nặng. Mặc dù nằm ở phổ đối diện của sự rối loạn về trọng lượng, phần lớn các gen ký ức phiên mã của tình trạng thiếu và thừa dinh dưỡng không trùng lặp, cho thấy các cơ chế khác nhau tham gia trong những trạng thái dinh dưỡng cực đoan này.

Herpes simplex type I (HSV)-based vectors have been used experimentally for suicide gene therapy, immunomodulatory gene delivery, and direct oncolytic therapy. The current study utilizes the novel concept of regional delivery of an oncolytic virus in combination with or serving as the helper virus for packaging herpes-based amplicon vectors carrying a cytokine transgene, with the goal of identifying if this combination is more efficacious than either modality alone. A replication competent oncolytic HSV (G207) and a replication incompetent HSV amplicon carrying the gene for the immunomodulatory cytokine IL-2 (HSV-IL2) were tested in murine syngeneic colorectal carcinoma and in rat hepatocellular carcinoma models. Liver tumors were treated with vascular delivery of (1) phosphate-buffered saline (PBS), (2) G207, (3) HSV-IL2, (4) G207 and HSV-IL2 mixed in combination (mG207/HSV-IL2), and (5) G207 as the helper virus for packaging the construct HSV-IL2 (pG207/HSV-IL2). Tumor burden was significantly reduced in all treatment groups in both rats and mice treated with highdose G207, HSV-IL2, or both (p < 0.02). When a low dose of virus was used in mice, anti-tumor efficacy was improved by use of G207 and HSV-IL2 in combination or with HSV-IL2 packaged by G207 (p < 0.001). This improvement was abolished when CD4+ and CD8+ lymphocytes were depleted, implying that the enhanced anti-tumor response to low-dose combined therapy is immune mediated. Vascular regional delivery of oncolytic and amplicon HSV vectors can be used to induce improved anti-tumor efficacy by combining oncolytic and immunostimulatory strategies.

Dendritic cell (DC)-based adoptive tumor immunotherapy approaches have shown promising results, but the incidence of tumor regression is low and there is an evident call for identifying culture conditions that produce DCs with a more potent Th1 potential. Routinely, DCs are differentiated in CO2 incubators under atmospheric oxygen conditions (21% O2), which differ from physiological oxygen levels of only 3–5% in tissue, where most DCs reside. We investigated whether differentiation and maturation of DCs under physiological oxygen levels could produce more potent T-cell stimulatory DCs for use in adoptive immunotherapy. We found that immature DCs differentiated under physiological oxygen levels showed a small but significant reduction in their endocytic capacity. The different oxygen levels did not influence their stimuli-induced upregulation of cluster of differentiation 54 (CD54), CD40, CD83, CD86, C-C chemokine receptor type 7 (CCR7), C-X-C chemokine receptor type 4 (CXCR4) and human leukocyte antigen (HLA)-DR or the secretion of interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-10 in response to lipopolysaccharide (LPS) or a cytokine cocktail. However, DCs differentiated under physiological oxygen level secreted higher levels of IL-12(p70) after exposure to LPS or CD40 ligand. Immature DCs differentiated at physiological oxygen levels caused increased T-cell proliferation, but no differences were observed for mature DCs with regard to T-cell activation. In conclusion, we show that although DCs generated under atmospheric or physiological oxygen conditions are mostly similar in function and phenotype, DCs differentiated under physiological oxygen secrete larger amounts of IL-12(p70). This result could have implications for the use of ex vivo-generated DCs for clinical studies, since DCs differentiated at physiological oxygen could induce increased Th1 responses in vivo.

B cell receptor Immunoglobulin (BcR IG) repertoire of Chronic Lymphocytic Leukemia (CLL) is characterized by the expression of quasi-identical BcR IG. These are observed in approximately 30% of patients, defined as stereotyped receptors and subdivided into subsets based on specific VH CDR3 aa motifs and phylogenetically related IGHV genes. Although relevant to CLL ontogeny, the distribution of CLL-biased stereotyped immunoglobulin rearrangements (CBS-IG) in normal B cells has not been so far specifically addressed using modern sequencing technologies. Here, we have investigated the presence of CBS-IG in splenic B cell subpopulations (s-BCS) and in CD5+ and CD5− B cells from the spleen and peripheral blood (PB). Fractionation of splenic B cells into 9 different B cell subsets and that of spleen and PB into CD5+ and CD5− cells were carried out by FACS sorting. cDNA sequences of BcR IG gene rearrangements were obtained by NGS. Identification of amino acidic motifs typical of CLL stereotyped subsets was carried out on IGHV1-carrying gene sequences and statistical evaluation has been subsequently performed to assess stereotypes distribution. CBS-IG represented the 0.26% average of IGHV1 genes expressing sequences, were detected in all of the BCS investigated. CBS-IG were more abundant in splenic and circulating CD5+ B (0.57%) cells compared to CD5− B cells (0.17%). In all instances, most CBS IG did not exhibit somatic hypermutation similar to CLL stereotyped receptors. However, compared to CLL, they exhibited a different CLL subset distribution and a broader utilization of the genes of the IGHV1 family. CBS-IG receptors appear to represent a part of the “public” BcR repertoire in normal B cells. This repertoire is observed in all BCS excluding the hypothesis that CLL stereotyped BcR accumulate in a specific B cell subset, potentially capable of originating a leukemic clone. The different relative representation of CBS-IG in normal B cell subgroups suggests the requirement for additional selective processes before a full transformation into CLL is achieved.

Calcific aortic valve disease (CAVD) is common among the elderly, and aortic valve interstitial cells (AVICs) exhibit unique inflammatory and osteogenic responses to proinflammatory stimulation, which plays an important role in valvular fibrosis and calcification. Thus, suppression of AVIC proinflammatory response may have therapeutic utility for the prevention of CAVD progression. Interleukin (IL)-37, an antiinflammatory cytokine, reduces tissue inflammation. This study aimed to test the hypothesis that IL-37 suppresses human AVIC inflammatory responses to Toll-like receptor (TLR) agonists. Human AVICs were exposed to Pam3CSK4, poly(I:C) and lipopolysaccharide in the presence and absence of recombinant human IL-37. Stimulation of TLR4 increased the production of intercellular adhesion molecule-1, IL-6, IL-8 and monocyte chemoattractant protein-1. Knockdown of myeloid differentiation factor 88 (MyD88) or TIR-domain-containing adaptor-inducing interferon-β differentially affected inflammatory mediator production following TLR4 stimulation. IL-37 reduced production of these inflammatory mediators induced by TLR4. Moreover, knockdown of IL-37 enhanced induction of these mediators by TLR4. IL-37 also suppressed inflammatory mediator production induced by the MyD88-dependent TLR2, but had no effect on the inflammatory responses to the TRIF-dependent TLR3. Furthermore, IL-37 inhibited NF-κB activation induced by TLR2 or TLR4 through a mechanism dependent of IL-18 receptor α-chain. Activation of TLR2, TLR3 or TLR4 upregulates the production of inflammatory mediators in human AVICs. IL-37 suppresses MyD88-mediated responses to reduce inflammatory mediator production following stimulation of TLR2 and TLR4. This antiinflammatory cytokine may be useful for suppression of aortic valve inflammation elicited by MyD88-dependent TLR signaling.

We recently analyzed the hypermethylation status of the p16INK4a (p16) gene promoter in normal-appearing mucosa obtained from patients with colorectal cancer. Hypermethylation of p16 was associated with reduced survival of these patients. In the present study, germ line polymorphisms in the folate- and methyl-associated genes, methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR) and methionine synthase reductase (MTRR), were analyzed in the same patient cohort to find a possible link between these genetic variants and p16 hypermethylation. Genomic DNA was extracted from blood of patients (n = 181) and controls (n = 300). Genotype analyses were run on an ABI PRISM® 7900HT sequence-detection system (Applied Biosystems), using real-time polymerase chain reaction and TaqMan chemistry. The results showed that the genotype distributions of the patient and control groups were similar. No significant differences in cancer-specific or disease-free survival of stage I–III patients according to polymorphic variants were detected, nor were any differences in cancer-specific or disease-free survival detected when patients were subgrouped according to the MTHFR or MTR genotype groups and dichotomized by p16 hypermethylation status in mucosa. However, patients with the MTRR 66 AA/AG genotypes were found to have a significantly worse cancer-specific survival when the mucosa were positive, compared with negative, for p16 hypermethylation (hazard ratio 2.7; 95% confidence interval 1.2–6.4; P = 0.023). In contrast, there was no difference in survival among patients with the MTRR 66 GG genotype stratified by p16 hypermethylation status. These results indicate a relationship between genetic germ-line variants of the MTRR gene and p16 hypermethylation in mucosa, which may affect the clinical outcome of patients with colorectal cancer.

Certain gene therapy protocols may require multiple administrations of vectors to achieve therapeutic benefit to the patient. This may be especially relevant for vectors such as adenoviral vectors that do not integrate into the host chromosome. Because immunocompetent animal models used for gene transfer studies develop neutralizing antibodies to adenoviral vectors after a single administration, little is known about how repeat administrations of vectors might affect transgene expression and vector toxicity. We used mice deficient in the membrane spanning region of immunoglobulin (IgM), which do not develop antibodies, to evaluate the effect of repeated intravenous administration of first-generation and helper-dependent adenoviral vectors expressing human α1-antitrypsin (hAAT). The duration and levels of transgene expression were evaluated after repeated administration of vectors. Toxicity was assessed by measuring the level of liver enzymes in the serum and the degrees of hepatocyte hypertrophy and proliferation. We found that previous administration of first-generation adenoviral vectors can alter the response to subsequent doses. These alterations included an increase in transgene expression early (within 1 and 3 days), followed by a rapid drop in expression by day 7. In addition, previous administrations of first-generation vectors led to an increase in toxicity of subsequent doses, as indicated by a rise in liver enzymes and an increase in hepatocyte proliferation. In contrast to first-generation vectors, use of the helper-dependent adenovirus vector, AdSTK109, which contained no viral coding regions, did not lead to increased toxicity after multiple administrations. We conclude that the response of the host to adenoviral vectors can be altered after repeated administration, compared with the response after the initial vector dose. In addition, these experiments provide further evidence for the relative safety of helper-dependent adenoviral vectors for gene therapy, compared with first-generation vectors.