Biomedical subjects

Changyu Zheng

Publications and source records attributed to Changyu Zheng.

16 recordsLinked to original sources

PubMed2025

GZMK+CD8+ T cells target a specific acinar cell type in Sjögren's disease.

OBJECTIVES: Sjögren's disease (SjD) is a systemic autoimmune disorder characterized by dysfunction of exocrine glands, particularly the salivary and lacrimal glands, with no clear etiology or effective therapy. This study explores the complex interplay of varied cell types in the salivary glands and their role in the pathology of Sjögren's disease. METHODS: Utilizing single-cell and spatial transcriptomics alongside spatial immunophenotyping to analyze human minor salivary glands, we developed a comprehensive understanding of the cellular landscape of non-SjD salivary glands and how that landscape changes in SjD patients. In vitro cellular assays and novel patient-derived primary epithelial cells were co-cultured with autologous T cells to confirm effector states and the delivery and effect of disease-associated granzymes. RESULTS: We identified previously unrecognized heterogeneity among acinar cells, including a PRR4⁺CST3⁺WFDC2⁻ seromucous acinar population that is selectively lost in Sjögren's disease. Expression and organizational changes were linked to clinical features: (i) T cells in the glands of SSA⁺, high-focus score patients showed increased transcriptional signatures of activation, antigen presentation, and apoptosis resistance compared with patients with mild or moderate disease, and (ii) patients with low immune infiltration exhibited distinct epithelial organization. Notably, GZMK⁺CD8⁺ T cells, which accumulate with disease severity, displayed a cytotoxic transcriptional program, degranulated upon stimulation ex vivo, and localized spatially with immune-engaged epithelial cells. Functional assays demonstrated that GZMK activates interferon signaling in vitro, and autologous co-cultures of patient-derived T cells and epithelial cells validated these findings. CONCLUSIONS: Using single-cell and spatial transcriptomics and proteomics, this study identifies a selective loss of PRR4⁺CST3⁺WFDC2⁻ seromucous acinar cells and a rise in GZMK⁺CD8⁺ T cells in Sjögren's disease, revealing distinct immune-mediated epithelial remodeling and interferon-driven dysfunction across diverse clinical presentations. These findings uncover a novel sub-cytolytic effector mechanism by which GZMK⁺CD8⁺ T cells impair mitochondrial integrity and activate innate immune signaling, linking epithelial injury to type I interferon responses and offering new therapeutic targets.

Humans↗

PubMed2006

Toxicity and biodistribution of a first-generation recombinant adenoviral vector, encoding aquaporin-1, after retroductal delivery to a single rat submandibular gland.

Before conducting a phase 1/2 clinical trial of a serotype 5 adenovirus encoding human aquaporin-1 (AdhAQP1) for the treatment of radiation-damaged salivary glands, we have conducted a detailed toxicity and biodistribution study in adult rats. AdhAQP1 (2x108-2x1011 particles) was delivered to a single submandibular gland by retroductal cannulation. Administration of this vector resulted in no animal mortality or morbidities, and no adverse signs of clinical toxicity. In addition, over the 92-day time course of the study, with both male and female rats, there were no consistent treatment-related changes in serum indicators of hepatic, renal, and cardiac functions. Importantly, we also observed no vector-associated effects on either water consumption by, or hematocrit levels in, study animals. However, three suggestive mild gender-related response differences were seen. Female, but not male, rats exhibited small reductions in food consumption (10-15%) and body weight gain (5-10%), and evidence of persistent inflammation, after vector treatment. These were vector, but not dose, related. Three days after delivery of 2x1011 particles of AdhAQP1, vector was detected primarily in the targeted gland; 9 of 10 samples from the targeted gland were positive, whereas only 5 of 90 nonoral samples were positive. There was no evidence of the generation of replication-competent adenovirus in saliva or blood samples. In aggregate, these findings show that localized delivery of AdhAQP1 to salivary glands appears to occur without significant toxicity.

Adenoviridae↗

PubMed2005

Transfer of the AQP1 cDNA for the correction of radiation-induced salivary hypofunction.

The treatment of most patients with head and neck cancer includes ionizing radiation (IR). Salivary glands in the IR field suffer significant and irreversible damage, leading to considerable morbidity. Previously, we reported that adenoviral (Ad)-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to rat [C. Delporte, B.C. O'Connell, X. He, H.E. Lancaster, A.C. O'Connell, P. Agre, B.J. Baum, Increased fluid secretion after adenoviral-mediated transfer of the aquaporin-1 cDNA to irradiated rat salivary glands. Proc. Natl. Acad. Sci. U S A. 94 (1997) 3268-3273] and miniature pig [Z. Shan, J. Li, C. Zheng, X. Liu, Z. Fan, C. Zhang, C.M. Goldsmith, R.B. Wellner, B.J Baum, S. Wang. Increased fluid secretion after adenoviral-mediated transfer of the human aquaporin-1 cDNA to irradiated miniature pig parotid glands. Mol. Ther. 11 (2005) 444-451] salivary glands approximately 16 weeks following IR resulted in a dose-dependent increase in salivary flow to > or =80% control levels on day 3. A control Ad vector was without any significant effect on salivary flow. Additionally, after administration of Ad vectors to salivary glands, no significant lasting effects were observed in multiple measured clinical chemistry and hematology values. Taken together, the findings show that localized delivery of AdhAQP1 to IR-damaged salivary glands is useful in transiently increasing salivary secretion in both small and large animal models, without significant general adverse events. Based on these results, we are developing a clinical trial to test if the hAQP1 cDNA transfer strategy will be clinically effective in restoring salivary flow in patients with IR-induced parotid hypofunction.

Animals↗

PubMed2005

Evaluation of viral and mammalian promoters for use in gene delivery to salivary glands.

To optimize vectors for salivary gland gene transfer, we screened viral [cytomegalovirus (CMV; human immediate early), Rous sarcoma virus (RSV), simian virus 40, and Moloney murine leukemia virus long terminal repeat] and mammalian [elongation factor 1alpha (EF1alpha), cytokeratin 18 (K18), cytokeratin 19 (K19), kallikrein (Kall), and amylase (AMY), all human, and rat aquaporin-5 (rAQP5), and derivative elements] promoters driving luciferase activity in vitro and in vivo. In adenoviral vectors, the CMV promoter showed highest activity, with the EF1alpha and RSV promoters slightly less powerful, in rat submandibular glands (SMGs). The K18 2.5-kb, K19 3.0-kb, and rAQP5 0.4-kb and Kall promoters had intermediate activity, while the AMY promoter exhibited lowest activity. To localize transgene expression, enhanced green fluorescence protein was used. The CMV, RSV, EF1alpha, K18 2.5-kb, K19 3.0-kb, rAQP5 0.4-kb, and AMY promoters were not cell-type specific in SMGs; however, the Kall promoter was primarily active in ductal cells. These data will facilitate optimal expression cassette design for salivary gland gene transfer.

Adenoviridae↗

PubMed2005

Intratumoral administration of immature dendritic cells following the adenovirus vector encoding CD40 ligand elicits significant regression of established myeloma.

Our previous study showed that J558 myeloma cells engineered CD40L lost their tumorigenicity in syngeneic mice, and the inoculation of J558/CD40L tumor cells further led to the protective immunity against wild tumors. In the present study, we investigated whether the vaccine can exert more efficient antitumor immunity by combination with adenovirus mediated CD40L gene therapy and immature dendritic cells (iDCs). The results demonstrated that intratumoral administration of iDCs 2 days after AdVCD40L injection, not only significantly suppressed the tumor growth, but also eradiated the established tumors in 40% of the mice. The potent antitumor effect produced by the combination therapy correlated with high expression of MHC, costimulatory and Fas molecules on J558 cells, which was derived from CD40L transgene expression. In addition, transgene CD40L expression could dramatically induce J558 cell apoptosis. Effectively capturing apoptotic bodies by iDCs in vivo could induce DC maturation, prime tumor-specific CTLs and tend to Th1-type immune response. Finally, in vivo depletion experimentation suggested both CD4+ and CD8+ T cells were involved in mediating the antitumor immune responses of combined treatment of AdVCD40L and iDCs, with CD8+ T cells being the major effector. These findings could be beneficial for designing strategies of DCs vaccine and CD40L for anticancer immunotherapy.

Adenoviridae↗

PubMed2005

Significant tumor regression induced by microencapsulation of recombinant tumor cells secreting fusion protein.

Implantation of microencapsulated engineered cells secreting molecules with antineoplastic properties into tumors is a novel approach to cancer gene therapy. In this study, we constructed an engineered tumor cell line, VkCk/RM4-TNF-alpha, which secreted RM4/TNF-alpha fusion protein containing the chimeric antitumor antibody, F(ab')2 (RM4), recognizing the tumor antigen TAG72, as well as the TNF-alpha moiety. The engineered cells were encapsulated into microencapsules. The RM4/TNF-alpha fusion protein secreted by encapsulated VkCk/RM4-TNF-alpha cells could be diffused through the microencapsule membrane into the supernatant and exert a cytotoxic effect on L929 cells in vitro. The antigen-specific binding-reactivity of RM4/TNF-alpha for the TAG72 antigen was confirmed by immunohistochemical staining of rat LMCR tumor cells which expressed TAG72 antigen. Implantation of microencapsules containing VkCk/RM4-TNF-alpha cells into LMCR tumors in rats induced tumor regression as a result of tumor necrosis formation. Taken together, these data suggest that microencapsulation of recombinant tumor cells secreting antibody/cytokine fusion protein might be an alternative approach in the treatment of cancers.

Animals↗

PubMed2005

Vaccine of engineered tumor cells secreting stromal cell-derived factor-1 induces T-cell dependent antitumor responses.

The CXC chemokine SDF-1 has been characterized as a T-cell chemoattractant both in vitro and in vivo. To determine whether SDF-1 expression within tumors can influence tumor growth, we transfected an expression vector pCI-SDF-1 for SDF-1 into J558 myeloma cells and tested their ability to form tumors in BALB/c. Production of biologically active SDF-1 (1.2 ng/mL) was detected in the culture supernatants of cells transfected with the expression vector pCI-SDF-1. J558 cells gave rise to a 100% tumor incidence, whereas SDF-1-expressing J558/SDF-1 tumors invariably regressed in BALB/c mice and became infiltrated with CD4(+) and CD8(+) T cells. Regression of the J558/SDF-1 tumors was dependent on both CD4(+) and CD8(+) T-cells. Our data also indicate that TIT cells containing both CD4(+) and CD8(+) T-cells within J558/SDF-1 tumors express the SDF-1 receptor CXCR4, and that SDF-1 specifically chemoattracts these cells in vitro. Furthermore, immunization of mice with engineered J558/SDF-1 cells elicited the most potent protective immunity against 0.5 x 10(6) cells J558 tumor challenge in vivo, compared to immunization with the J558 alone, and this antitumor immunity mediated by J558/SDF-1 tumor cell vaccination in vivo appeared to be dependent on CD8(+) CTL. Thus, SDF-1 has natural adjuvant activities that may augment antitumor responses through their effects on T-cells and thereby could be important in gene transfer immunotherapies for some cancers.

Animals↗

PubMed2005

Salivary glands as a potential gene transfer target for gene therapeutics of some monogenetic endocrine disorders.

Salivary glands (SGs) exhibit several important features which are also common to endocrine glands: self-containment due to a surrounding capsule, highly efficient protein production and the ability to secrete proteins into the bloodstream. We have hypothesized that SGs are potentially useful as gene transfer targets for the correction of inherited monogenetic endocrine disorders. In the present communication, we extend our studies and attempt to test our hypothesis by comparing the efficacy of two commonly used viral vectors and the resulting serum and salivary distribution of transgene encoded hormones. A low dose (5 x10(9) particles) of either an adenoviral serotype 5 (Ad5) vector encoding the human erythropoietin (hEPO) cDNA or an adeno-associated virus sero-type 2 (AAV2) vector encoding either the hEPO or human growth hormone (hGH) cDNA was administered to individual submandibular SGs of Balb/c mice. Serum and salivary hEPO and hGH levels were determined at defined time points. Two additional recombinant viruses encoding enhanced green fluorescence protein (GFP) were also used (AdGFP and AAVGFP) in order to perform immunohistochemical analyses of transgenic protein localization in SG sections post-administration. AAV2 vectors led to stable gene transfer unlike the results with the Ad5 vectors. Indeed, in one mouse we observed hEPO production for a period of two years after administration of AAVhEPO to SGs. hEPO, which is a constitutive pathway secretory protein, was readily secreted into the bloodstream from the SGs, yielding therapeutically adequate serum levels. Conversely, hGH, a regulated secretory pathway protein, was preferentially secreted into saliva. SGs may be an attractive candidate target tissue for gene therapeutics of some monogenetic endocrine deficiency disorders. At present, AAV2 vectors seem particularly useful for such applications, and transgenes encoding constitutive secretory pathway hormones are more suitable for this application with SGs than those encoding regulated secretory pathway hormones.

Adenoviridae↗

PubMed2004

Reengineered salivary glands are stable endogenous bioreactors for systemic gene therapeutics.

The use of critical-for-life organs (e.g., liver or lung) for systemic gene therapeutics can lead to serious safety concerns. To circumvent such issues, we have considered salivary glands (SGs) as an alternative gene therapeutics target tissue. Given the high secretory abilities of SGs, we hypothesized that administration of low doses of recombinant adeno-associated virus (AAV) vectors would allow for therapeutic levels of transgene-encoded secretory proteins in the bloodstream. We administered 10(9) particles of an AAV vector encoding human erythropoietin (hEPO) directly to individual mouse submandibular SGs. Serum hEPO reached maximum levels 8-12 weeks after gene delivery and remained relatively stable for 54 weeks (longest time studied). Hematocrit levels were similarly increased. Moreover, these effects proved to be vector dose-dependent, and even a dosage as low as 10(8) particles per animal led to significant increases in hEPO and hematocrit levels. Vector DNA was detected only within the targeted SGs, and levels of AAV copies within SGs were highly correlated with serum hEPO levels (r = 0.98). These results show that SGs appear to be promising targets with potential clinical applicability for systemic gene therapeutics.

Animals↗

PubMed2004

Combined alpha tumor necrosis factor gene therapy and engineered dendritic cell vaccine in combating well-established tumors.

BACKGROUND: Although current immunotherapeutic strategies including adenovirus (AdV)-mediated gene therapy and dendritic cell (DC) vaccine can all stimulate antitumor cytotoxic T lymphocyte (CLT) responses, their therapeutic efficiency has still been limited to generation of prophylactic antitumor immunity against re-challenge with the parental tumor cells or growth inhibition of small tumors in vivo. However, it is the well-established tumors in animal models that mimic clinical patients with existing tumor burdens. Alpha tumor necrosis factor (TNF-alpha) is a multifunctional and immunoregulatory cytokine that induces antitumor activity and activates immune cells such as DCs and T cells. We hypothesized that a combined immunotherapy including gene therapy and DC vaccine would have some advantages over each modality administered as a monotherapy. METHODS: We investigated the antitumor immunotherapeutic efficiency of gene therapy by intratumoral injection of AdVTNF-alpha and DC vaccine using subcutaneous injection of TNF-alpha-gene-engineered DC(TNF-alpha) cells, and further developed a combined AdV-mediated TNF-alpha-gene therapy and TNF-alpha-gene-engineered DC(TNF-alpha) vaccine in combating well-established MO4 tumors expressing the ovalbumin (OVA) gene in an animal model. RESULTS: Our data show that vaccination of DC(TNF-alpha) cells pulsed with the OVA I peptide can (i) stimulate type 1 immune response with enhanced antitumor CTL activities, (ii) induce protective immunity against challenge of 5 x 10(5) MO4 tumor cells, and (iii) reduce growth of the small (3-4 mm in diameter), but not large, established MO4 tumors (6-8 mm in diameter). Our data also show that AdVTNF-alpha-mediated gene therapy can completely eradicate small tumors in 6 out of 8 (75%) mice due to the extensive tumor necrosis formation, but not the large tumors (0%). Interestingly, a combined AdVTNF-alpha-mediated gene therapy and TNF-alpha-gene-engineered DC(TNF-alpha) vaccine is able to cure 3 out of 8 (38%) mice bearing large MO4 tumors, indicating that the combined immunotherapy strategy is much more efficient in combating well-established tumors than monotherapy of either gene therapy or DC vaccine alone. CONCLUSIONS: This novel combined immunotherapy may become a tool of considerable conceptual interest in the implementation of future clinical objectives.

Animals↗

PubMed2003

Advances in vector-mediated gene transfer.

Clinical applications of gene transfer technology initially targeted the treatment of inherited monogenetic disorders and cancers refractory to conventional therapies. Today, gene transfer approaches are being developed for most tissues and for multiple disorders including those affecting quality of life. The focus herein is eventual application of gene transfer technology for the management of organ-directed autoimmunity. A specific example is presented: Sjögren's syndrome and localized salivary gland gene transfer. The status of relevant pre-clinical gene transfer studies is reviewed, with an emphasis on use of adenoviral and adeno-associated viral vectors. Current limitations of effective organ-directed gene transfer are also discussed.

Adenoviridae↗

PubMed2003

Inclusion of Moloney murine leukemia virus elements upstream of the transgene cassette in an E1-deleted adenovirus leads to an unusual genomic integration in epithelial cells.

Classically, the 5' and 3' long terminal repeats (LTRs) are considered necessary but not sufficient for retroviral integration. Recently, we reported that inclusion of these and additional elements from Moloney murine leukemia virus (MoMLV) facilitated transgene integration, without retroviral integrase, when placed in an adenoviral context (AdLTR-luc vector) (Nat. Biotech. 18 (2000), 176; Biochem. Biophys. Res. Commun. 300 (2003), 115). To help understand this nonhomologous DNA recombination event, we constructed another vector, AdELP-luc, with 2.7 kb of MoMLV elements identically placed into an E1-deleted adenovirus type 5 backbone upstream of a luciferase cDNA reporter gene. Unlike AdLTR-luc, no MoMLV elements were placed downstream of the expression cassette. AdELP-luc readily infected epithelial cells in vitro. Southern hybridizations with DNA from cloned cells showed that disruption of the MoMLV sequences occurred. One cell clone, grown in vitro without any special selection medium for 9 months, exhibited stable vector integration and luciferase activity. Importantly, both Southern hybridization and FISH analyses showed that in addition to the MoMLV elements and expression cassette, substantial adenoviral sequence downstream of the luciferase cDNA was genomically integrated. These results suggest that the 2.7 kb of MoMLV sequence included in AdELP-luc have cis-acting functions and mediates an unusual integration event.

Adenoviridae↗

PubMed2003

Integration efficiency of a hybrid adenoretroviral vector.

The frequency with which the hybrid vector AdLTR-luc mediates genomic integration [Nat. Biotech. 18 (2000) 176-180] is unknown. To address this, we constructed AdLTR-red, using the AdLTR-luc backbone and the enhanced red fluorescence protein cDNA. Kinetic studies showed that AdLTR-red and a conventional adenoviral vector, AdCMV-red, entered and transduced epithelial cells comparably. AdLTR-red integration frequency in vitro, i.e., the percentage of red clones after 10-12 doubling times from limiting dilutions, was 8.0% (36/450; at 67 particles/cell). With AdCMV-red, 0/549 clones were integration-positive. Seven days after AdLTR-luc or AdCMV-luc (10(11) particles) femoral vein administration to adult rats splenocytes were prepared, stimulated with concanavalin A, and examined by FISH. AdLTR-luc integration occurred in 5-11% of mitotic rat splenocytes, while no unequivocal integration was found with AdCMV-luc. These data provide the first quantitative evidence of the frequency for genomic integration with this hybrid vector.

Adenoviridae↗

PubMed2003

Distribution and toxicity resulting from adenoviral vector administration to a single salivary gland in adult rats.

BACKGROUND: We examined the distribution and toxicity associated with a single salivary gland administration of a recombinant adenoviral vector, AdCMVH3, encoding human histatin 3. METHODS: Adult rats received different doses of AdCMVH3 (0, 106, 3 x 107, and 109 pfu; 50 microl) via the right submandibular gland and were followed for 15 days. Food consumption, weight gain, clinical appearance, and serum chemistry were monitored, and a necropsy was performed. Vector distribution was examined by polymerase chain reaction, and selected saliva samples were tested for replication-competent adenovirus (RCA). RESULTS: All animals survived to sacrifice (days 2, 8, and 15), and appeared normal clinically. There were no differences in food consumption, weight gain, and serum chemistry. The only consistent necropsy findings were lymphoid infiltrates and necrosis in the target submandibular glands of high-dosage animals. AdCMVH3 detection was virus dose dependent, decreased with time, and at low dose preferentially observed in the targeted gland. No RCA was detected. CONCLUSIONS: Salivary gland administration of 109 pfu AdCMVH3 elicits an initial focal pathologic response and wide tissue distribution. There is no associated systemic toxicity up to 15 days, and lower doses are primarily found in glands.

Adenoviridae↗

PubMed2002

Long-term expression after infection by the hybrid vector AdLTR-luc is from integrated transgene.

The novel adenoretroviral vector, AdLTR-luc, infects dividing and nondividing cells, and mediates long-term transgene expression(Zheng, C., Baum, B. J., Iadarola, M. J., and O'Connell, B. C., Nat. Biotech. 18, 176-180, 2000). To determine the source of this expression we examined two epithelial cell lines. One, HSG, permits E1(-) recombinant adenoviral replication, while the other, A5, does not. An HSG clone, that expressed luciferase stably for > 6 months, was obtained following infection at approximately 0.2 AdLTR-luc particles/cell. Southern and PCR analyses showed that luciferase cDNA present was integrated. A5 cells were infected with AdLTR-luc at approximately 1000 particles/cell, and colonies were obtained by limiting dilution. Eight clones showed stable luciferase activity for > 9 months. High molecular weight DNA extracts from clones were positive for genomic integration by Southern, PCR, and quantitative PCR analyses. Similar analyses of low molecular weight DNA extracts indicated the absence of intact extrachromosomal vector. These data demonstrate that long-term luciferase expression after infection by AdLTR-luc is derived from the integrated cDNA.

Adenoviridae↗

PubMed2002

Gene transfer to salivary glands.

This article provides a review of the application of gene transfer technology to studies of salivary glands. Salivary glands provide an uncommon target site for gene transfer but offer many experimental situations likely of interest to the cell biologist. The reader is provided with a concise overview of salivary biology, along with a general discussion of the strategies available for gene transfer to any tissue. In particular, adenoviral vectors have been useful for proof of concept studies with salivary glands. Several examples are given, using adenoviral-mediated gene transfer, for addressing both biological and clinical questions. Additionally, benefits and shortcomings affecting the utility of this technology are discussed.

Animals↗