Liver Sinusoidal Endothelial Cells Are a Site of Murine Cytomegalovirus Latency and Reactivation

Latent cytomegalovirus (CMV) is frequently transmitted by organ transplantation, and its reactivation under conditions of immunosuppressive prophylaxis against graft rejection by host-versus-graft disease bears a risk of graft failure due to viral pathogenesis. CMV is the most common cause of infection following liver transplantation. Although hematopoietic cells of the myeloid lineage are a recognized source of latent CMV, the cellular sites of latency in the liver are not comprehensively typed. Here we have used the BALB/c mouse model of murine CMV infection to identify latently infected hepatic cell types. We performed sex-mismatched bone marrow transplantation with male donors and female recipients to generate latently infected sex chromosome chimeras, allowing us to distinguish between Y-chromosome (gene sry or tdy)-positive donor-derived hematopoietic descendants and Y-chromosome-negative cells of recipients'' tissues. The viral genome was found to localize primarily to sry-negative CD11b⁻ CD11c⁻ CD31⁺ CD146⁺ cells lacking major histocompatibility complex class II antigen (MHC-II) but expressing murine L-SIGN. This cell surface phenotype is typical of liver sinusoidal endothelial cells (LSECs). Notably, sry-positive CD146⁺ cells were distinguished by the expression of MHC-II and did not harbor latent viral DNA. In this model, the frequency of latently infected cells was found to be 1 to 2 per 10⁴ LSECs, with an average copy number of 9 (range, 4 to 17) viral genomes. Ex vivo-isolated, latently infected LSECs expressed the viral genes m123/ie1 and M122/ie3 but not M112-M113/e1, M55/gB, or M86/MCP. Importantly, in an LSEC transfer model, infectious virus reactivated from recipients'' tissue explants with an incidence of one reactivation per 1,000 viral-genome-carrying LSECs. These findings identified LSECs as the main cellular site of murine CMV latency and reactivation in the liver.In human cytomegalovirus (hCMV) infection, hematopoietic progenitor cells of the myeloid differentiation lineage are a recognized cellular site of virus latency (for more-recent reviews, see references 75 and 94), and cell differentiation-dependent as well as cytokine-mediated viral gene desilencing by chromatin remodeling is discussed as the triggering event leading to virus reactivation (for a review, see reference 7). Although hematopoietic stem cell or bone marrow transplantation (BMT) is frequently associated with hCMV reactivation and recurrence in recipients after hematoablative leukemia/lymphoma therapy, the incidence of virus recurrence and disease is highest in the combination of an hCMV-negative donor (D⁻) and an hCMV-positive recipient (R⁺) (D⁻R⁺ > D⁺R⁺ > D⁺R⁻), indicating that donor hematopoietic cells are not the only source of latent hCMV and actually not the predominant source (34). Rather, the recipients experience reactivation of their own virus. Just the opposite is true in the case of solid organ transplantation, where the reactivating virus is mostly transmitted with the transplanted organ (D⁺R⁻ > D⁺R⁺ > D⁻R⁺) (34). Collectively, these risk assessments support the suggestion that reactivation, in both instances, occurs in latently infected tissue cells, that is, within the recipient''s organs and the transplanted donor organ, respectively. Although tissue-resident cells of hematopoietic origin remain candidates, stromal and parenchymal tissue cells come into consideration as additional sites of CMV latency.Longitudinal analysis of viral genome load in the latency models of murine CMV (mCMV) infection of neonatal mice (9, 91) as well as of adult mice after experimental BMT (8, 62, 64) has demonstrated a high viral latency burden in multiple organs long after clearance of viral DNA from bone marrow and blood (reviewed in reference 92). These findings support the suggestion that there exist two types of latency, namely, a temporary latency in hematopoietic cells and a latency in tissue cells that lasts through life. Accordingly, both types of latency may coexist early after primary infection, while “late latency” is restricted to organ sites. As we have shown previously in a sex-mismatched murine BMT model, bone marrow cells (BMCs) derived from latently infected donors in the phase of organ-restricted “late latency” cannot transmit latent or reactivated infection to naïve recipients upon intravenous cell transfer (99).A first hint for mCMV latency in stromal or reticular cells was presented long ago by Mercer and colleagues (73), who showed that infected cells during acute infection of the spleen are predominantly sinusoidal lining cells and that latent mCMV can be recovered from a major histocompatibility complex class II (MHC-II) antigen-negative and Thy-1 (CD90)-negative “stromal” cell fraction, which includes endothelial cells (ECs). These findings strongly argued against T and B lymphocytes, macrophages, and dendritic cells (DCs) being major reservoirs of latent mCMV in the spleen, a conclusion supported by later work of Pomeroy and colleagues (86). Similarly, Klotman and colleagues (54) as well as Hamilton and Seaworth (44) concluded that in kidney transplantation, donor kidney is the source of latent mCMV and that the latent viral genome is harbored by renal peritubular epithelial cells (53). A first hint for mCMV latency in ECs within the liver was provided by in situ PCR images presented by Koffron and colleagues (59) showing nuclear staining in cells with a microanatomical localization suspicious of liver sinusoidal ECs (LSECs). For hCMV, ECs, in particular those in arterial vessel walls, are regarded as a site of latency on the basis of the presence of viral DNA in cells expressing an EC marker (81; for a review, see reference 48), although other authors did not detect viral DNA in venous vessel walls (95). As discussed by Jarvis and Nelson (48), these data are not necessarily conflicting but may rather reflect the diversity of EC subsets at different anatomical locations (21, 27). As far as we know, and reactivation of productive infection from ECs that carry latent viral DNA is not yet formally proven for any type of EC.Hepatitis is a relevant organ manifestation of CMV disease in immunocompromised hosts (65), and hCMV reactivation has been reported to be the most common cause of infection following liver transplantation, in particular in a D⁺R⁻ combination (34, 76). In the murine model of immunocompromised hosts, viral histopathology in the liver is dominated by the cytopathogenic infection of hepatocytes, leading to extended plaque-like tissue lesions (41, 84; reviewed in reference 45). Occasionally, however, in these studies, infected hepatic ECs as well as Kupffer macrophages were detected by virus-specific immunohistology or by in situ virus-specific DNA hybridization.Using cell-type-specific conditional recombination of a fluorescence-tagged reporter virus in Cre-transgenic mice expressing Cre selectively in hepatocytes under the control of the albumin promoter, hepatocytes were recently identified as the main virus-producing cell type during mCMV infection. In Cre-transgenic mice expressing Cre selectively in vascular ECs under the control of the Tie2 promoter, the reporter virus was found to recombine also in LSECs, which released an amount of virus sufficient for virus spread to neighboring hepatocytes, although the virus productivity of LSECs was low and contributed little to the overall virus load in the liver (97).LSECs represent a unique liver-resident population of antigen-presenting cells that bear the capacity to cross-present antigens to naïve CD8 T cells (68, 69; reviewed in reference 57). They constitute the fenestrated endothelial lining of the hepatic sinusoids (15). By separating the sinusoidal compartment of the liver from the space of Disse and the liver parenchyma, LSECs form a boundary surface for sensing of pathogens and interaction with passenger lymphocytes. They perform a scavenger function contributing to hepatic clearance of bacterial degradation products derived from the gastrointestinal tract (103; reviewed in reference 57). According to this physiological role, antigen presentation by LSECs is associated with tolerance induction rather than with triggering an inflammatory immune response (29, 56, 68, 104).Here we provide evidence to support the suggestion that mCMV has chosen the tolerogenic and long-lived LSECs as an immunoprivileged niche for establishing viral latency in the liver.     

Adenosine Deaminase Enhances the Immunogenicity of Human Dendritic Cells from Healthy and HIV-Infected Individuals

ADA is an enzyme implicated in purine metabolism, and is critical to ensure normal immune function. Its congenital deficit leads to severe combined immunodeficiency (SCID). ADA binding to adenosine receptors on dendritic cell surface enables T-cell costimulation through CD26 crosslinking, which enhances T-cell activation and proliferation. Despite a large body of work on the actions of the ecto-enzyme ADA on T-cell activation, questions arise on whether ADA can also modulate dendritic cell maturation. To this end we investigated the effects of ADA on human monocyte derived dendritic cell biology. Our results show that both the enzymatic and non-enzymatic activities of ADA are implicated in the enhancement of CD80, CD83, CD86, CD40 and CCR7 expression on immature dendritic cells from healthy and HIV-infected individuals. These ADA-mediated increases in CD83 and costimulatory molecule expression is concomitant to an enhanced IL-12, IL-6, TNF-α, CXCL8(IL-8), CCL3(MIP1-α), CCL4(MIP-1β) and CCL5(RANTES) cytokine/chemokine secretion both in healthy and HIV-infected individuals and to an altered apoptotic death in cells from HIV-infected individuals. Consistently, ADA-mediated actions on iDCs are able to enhance allogeneic CD4 and CD8-T-cell proliferation, globally yielding increased iDC immunogenicity. Taken together, these findings suggest that ADA would promote enhanced and correctly polarized T-cell responses in strategies targeting asymptomatic HIV-infected individuals.     

Human Trophoblast Cells Are Permissive to the Complete Replicative Cycle of Human Cytomegalovirus

Human trophoblast cells were permissively infected by human cytomegalovirus. The kinetics of viral immediate-early, early, and late gene expression was clearly delayed compared to that in fibroblasts. Productive infection was unequivocally proven by the detection of virion particles, infectious virus in trophoblast culture supernatant, and cell-to-cell spread of cytomegalovirus from infected trophoblasts to uninfected fibroblasts. These observations indicate that infected trophoblasts may be involved in maternofetal transmission of human cytomegalovirus.     

A Synthetic Model of Human Beta-Thalassemia Erythropoiesis Using CD34+ Cells from Healthy Adult Donors

Based upon the lack of clinical samples available for research in many laboratories worldwide, a significant gap exists between basic and clinical studies of beta-thalassemia major. To bridge this gap, we developed an artificially engineered model for human beta thalassemia by knocking down beta-globin gene and protein expression in cultured CD34+ cells obtained from healthy adults. Lentiviral-mediated transduction of beta-globin shRNA (beta-KD) caused imbalanced globin chain production. Beta-globin mRNA was reduced by 90% compared to controls, while alpha-globin mRNA levels were maintained. HPLC analyses revealed a 96% reduction in HbA with only a minor increase in HbF. During the terminal phases of differentiation (culture days 14–21), beta-KD cells demonstrated increased levels of insoluble alpha-globin, as well as activated caspase-3. The majority of the beta-KD cells underwent apoptosis around the polychromatophilic stage of maturation. GDF15, a marker of ineffective erythropoiesis in humans with thalassemia, was significantly increased in the culture supernatants from the beta-KD cells. Knockdown of beta-globin expression in cultured primary human erythroblasts provides a robust ex vivo model for beta-thalassemia.     

Human Plasmacytoid Dendritic Cells Sense Lymphocytic Choriomeningitis Virus-Infected Cells In Vitro

We previously reported that exosomal transfer of hepatitis C virus (HCV) positive-strand RNA from human Huh-7 hepatoma cells to human plasmacytoid dendritic cells (pDCs) triggers pDC alpha/beta interferon (IFN-α/β) production in a Toll-like receptor 7 (TLR7)-dependent, virus-independent manner. Here we show that human pDCs are also activated by a TLR7-dependent, virus-independent, exosomal RNA transfer mechanism by human and mouse hepatoma and nonhepatoma cells that replicate the negative-strand lymphocytic choriomeningitis virus (LCMV).     

用PCR法检测献血员单个核细胞中的CMV—DNA

107份自愿献血新鲜血标本和22份库存血标本分别用PCR检测单个核细胞中巨细胞病毒DNA（CMV－DNA）和用ELISA检测其血浆中CMV－IgG。结果CMV－DNA阳性率达80．4％和77．3％,CMV－IgC阳性纺为65．9％。其中CMV－IgG阳性者,基本上都携带CMV－DNA;CMV－IgG阴性者,亦有部分携带CMV－DNA。因此认为献血员血液中CMV高带毒率应引起临床有关部门的高度重视;PCR是筛选无传播CMV危险性血液制品的最可靠方法。     

Later Passages of Neural Progenitor Cells from Neonatal Brain Are More Permissive for Human Cytomegalovirus Infection

Congenital human cytomegalovirus (HCMV) infection is the most frequent infectious cause of birth defects, primarily neurological disorders. Neural progenitor/stem cells (NPCs) are the major cell type in the subventricular zone and are susceptible to HCMV infection. In culture, the differentiation status of NPCs may change with passage, which in turn may alter susceptibility to virus infection. Previously, only early-passage (i.e., prior to passage 9) NPCs were studied and shown to be permissive to HCMV infection. In this study, NPC cultures derived at different gestational ages were evaluated after short (passages 3 to 6) and extended (passages 11 to 20) in vitro passages for biological and virological parameters (i.e., cell morphology, expression of NPC markers and HCMV receptors, viral entry efficiency, viral gene expression, virus-induced cytopathic effect, and release of infectious progeny). These parameters were not significantly influenced by the gestational age of the source tissues. However, extended-passage cultures showed evidence of initiation of differentiation, increased viral entry, and more efficient production of infectious progeny. These results confirm that NPCs are fully permissive for HCMV infection and that extended-passage NPCs initiate differentiation and are more permissive for HCMV infection. Later-passage NPCs being differentiated and more permissive for HCMV infection suggest that HCMV infection in fetal brain may cause more neural cell loss and give rise to severe neurological disabilities with advancing brain development.     

Type III IFNs Are Produced by and Stimulate Human Plasmacytoid Dendritic Cells

Plasmacytoid dendritic cells (pDC) are rare cells found in peripheral blood and lymphoid tissues. pDC are considered to be "professional" type I IFN-producing cells and produce 10- to 100-fold more IFN-α than other cell types in response to enveloped viruses or synthetic TLR7 and TLR9 agonists. In this study, purified pDC were found to express high levels of IFN-λ receptor mRNA, as well as cell-surface IFN-λ receptor. We have developed intracellular flow cytometry assays using Abs to IFN-λ1/3 or -λ2 to assess the expression of IFN-λ proteins by pDC. We observed that a subset of human pDC expresses only intracellular IFN-α, whereas another subset produces both IFN-α and IFN-λ after stimulation with virus or the TLR9 agonist, CpG A; the cells that coexpressed IFN-α and IFN-λ were the cells with the highest levels of IFN-α expression. Ab cross-linking of CD4 or CD303 molecules on pDC inhibited both HSV-induced IFN-λ and IFN-α production. Like the production of IFN-α, the HSV-induced IFN-λ production in pDC was mediated through TLR9 and independent of virus replication. Exogenous IFN-λ treatment of pDC resulted in increased virus-induced expression of both IFN-α and IFN-λ. In addition, both exogenous IFN-λ and -α inhibited dexamethasone-induced apoptosis of pDC. We conclude that pDC are major producers of IFN-λ1 and -λ2 in response to viral stimulation and also express functional receptors for this cytokine. Thus, IFN-λ can serve as an autocrine signal to strengthen the antiviral response of pDC by increasing IFN-α and IFN-λ production, resulting in prolonged pDC survival.     

Subviral Dense Bodies of Human Cytomegalovirus Stimulate Maturation and Activation of Monocyte-Derived Immature Dendritic Cells

Dendritic cells play a central role in the immune control of human cytomegalovirus (HCMV) infection. This work aimed at investigating the impact of noninfectious, subviral dense bodies of HCMV on the maturation and activation of dendritic cells (DC). Treatment of immature DC with dense bodies led to the maturation of these cells and significantly increased their capacity for cytokine release and antigen presentation. Dense body-activated DC may thereby contribute to the development of antiviral immunity.     

Critical Role of MDA5 in the Interferon Response Induced by Human Metapneumovirus Infection in Dendritic Cells and In Vivo

Human metapneumovirus (hMPV) is a respiratory paramyxovirus of global clinical relevance. Despite the substantial knowledge generated during the last 10 years about hMPV infection, information regarding the activation of the immune response against this virus remains largely unknown. In this study, we demonstrated that the helicase melanoma differentiation-associated gene 5 (MDA5) is essential to induce the interferon response after hMPV infection in human and mouse dendritic cells as well as in an experimental mouse model of infection. Our findings in vitro and in vivo showed that MDA5 is required for the expression and activation of interferon (IFN) regulatory factors (IRFs). hMPV infection induces activation of IRF-3, and it regulates the expression of IRF-7. However, both IRF-3 and IRF-7 are critical for the production of type I and type III IFNs. In addition, our in vivo studies in hMPV-infected mice indicated that MDA5 alters viral clearance, enhances disease severity and pulmonary inflammation, and regulates the production of cytokines and chemokines in response to hMPV. These findings are relevant for a better understanding of the pathogenesis of hMPV infection.     

Human Cytomegalovirus (HCMV)-Specific CD4+ and CD8+ T Cells Are Both Required for Prevention of HCMV Disease in Seropositive Solid-Organ Transplant Recipients

In solid-organ transplant recipients (SOTR) the protective role of human cytomegalovirus (HCMV)-specific CD4⁺, CD8⁺ and γδ T-cells vs. HCMV reactivation requires better definition. The aim of this study was to investigate the relevant role of HCMV-specific CD4⁺, CD8⁺ and γδ T-cells in different clinical presentations during the post-transplant period. Thirty-nine SOTR underwent virologic and immunologic follow-up for about 1 year after transplantation. Viral load was determined by real-time PCR, while immunologic monitoring was performed by measuring HCMV-specific CD4⁺ and CD8⁺ T cells (following stimulation with autologous HCMV-infected dendritic cells) and γδ T-cells by flow cytometry. Seven patients had no infection and 14 had a controlled infection, while both groups maintained CD4⁺ T-cell numbers above the established cut-off (0.4 cell/µL blood). Of the remaining patients, 9 controlled the infection temporarily in the presence of HCMV-specific CD8⁺ only, until CD4⁺ T-cell appearance; while 9 had to be treated preemptively due to a viral load greater than the established cut-off (3×10⁵ DNA copies/mL blood) in the absence of specific CD4⁺ T-cells. Polyfunctional CD8⁺ T-cells as well as Vδ2⁻ γδ T-cells were not associated with control of infection. In conclusion, in the absence of HCMV-specific CD4⁺ T-cells, no long-term protection is conferred to SOTR by either HCMV-specific CD8⁺ T-cells alone or Vδ2⁻ γδ T-cell expansion.     

Circadian Rhythms of Circulating NK Cells in Healthy and Human Immunodeficiency Virus-Infected Men

Antiviral immunity involves NK cells, which circulate rhythmically every 24 hours. We have investigated circadian and 12-hour rhythms in the peripheral count of circulating NK cells in 15 men infected with human immunodeficiency virus (HIV) and 13 healthy controls. We analyzed three phenotypes using double-labeling with monoclonal antibodies and flow cytometry assessment: CD3^? CD16⁺, CD3^?CD57⁺, and CD2⁺CD3^?. A statistical validation of time-dependent differences was achieved if significance (p < 0.05) was validated both with analysis of variance and cosinor. The circadian rhythm had a similar asymmetric waveform for the three phenotypes and is homogeneous on an individual basis. The circulating NK cell count peaked in the early morning and was low at night. A circadian rhythm and a circahemidian harmonic characterized all phenotypes in healthy subjects. We considered two groups of HIV-infected men: those who were asymptomatic (eight) and those with acquired immune deficiency syndrome (AIDS) (seven). Circadian changes in NK cell count were similar in both subgroups and in healthy controls. The circadian pattern was also consistent among individual patients. Asymptomatic HIV-infected men (early-stage disease) exhibited more pronounced 12-hour rhythmicity than did patients with AIDS or controls. The circulation of NK cells does not appear to share the same synchro-nizer(s) as other circulating T- or B-lymphocyte subsets. Thus, HIV infection gradually abolished circadian rhythmicity in circulating T and B cells, whereas it did not disturb that in NK cells.     

Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo

There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed.     

In Vivo Long-Term Monitoring of Circulating Tumor Cells Fluctuation during Medical Interventions

The goal of this research was to study the long-term impact of medical interventions on circulating tumor cell (CTC) dynamics. We have explored whether tumor compression, punch biopsy or tumor resection cause dissemination of CTCs into peripheral blood circulation using in vivo fluorescent flow cytometry and breast cancer-bearing mouse model inoculated with MDA-MB-231-Luc2-GFP cells in the mammary gland. Two weeks after tumor inoculation, three groups of mice were the subject of the following interventions: (1) tumor compression for 15 minutes using 400 g weight to approximate the pressure during mammography; (2) punch biopsy; or (3) surgery. The CTC dynamics were determined before, during and six weeks after these interventions. An additional group of tumor-bearing mice was used as control and did not receive an intervention. The CTC dynamics in all mice were monitored weekly for eight weeks after tumor inoculation. We determined that tumor compression did not significantly affect CTC dynamics, either during the procedure itself (P = 0.28), or during the 6-week follow-up. In the punch biopsy group, we observed a significant increase in CTC immediately after the biopsy (P = 0.02), and the rate stayed elevated up to six weeks after the procedure in comparison to the tumor control group. The CTCs in the group of mice that received a tumor resection disappeared immediately after the surgery (P = 0.03). However, CTC recurrence in small numbers was detected during six weeks after the surgery. In the future, to prevent these side effects of medical interventions, the defined dynamics of intervention-induced CTCs may be used as a basis for initiation of aggressive anti-CTC therapy at time-points of increasing CTC number.     

In Vitro Characterization of Circulating Endothelial Progenitor Cells Isolated from Patients with Acute Coronary Syndrome

Background

The current understanding of the functional characteristics of circulating endothelial progenitor cells (EPC) is limited, especially in patients affected by cardiovascular diseases. In this study, we have analyzed the in vitro clonogenic capacity of circulating EPC, also known as endothelial colony-forming cells (ECFC), in patients with acute coronary syndrome (ACS), in comparison to the colony forming unit-endothelial-like cells (CFU-EC) of hematopoietic/monocytic origin.

Methodology/Principal Findings

By culturing peripheral blood mononuclear cells (PBMC) of patients with ACS (n = 70), CFU-EC were frequently isolated (from 77% of ACS patients), while EPC/ECFC were obtained only in a small subset (13%) of PBMC samples, all harvested between 7–14 days after the acute cardiovascular event. Notably, ex-vivo generation of EPC/ECFC was correlated to a higher in vitro release of PDGF-AA by the corresponding ACS patient PBMC. By using specific endothelial culture media, EPC/ECFC displayed in vitro expansion capacity, allowing the phenotypic and functional characterization of the cells. Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: i) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, i) variable expression of the CD34, CD106 and CD184 markers, and iii) negativity for CD45, CD90, CD117 and CD133. Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities.

Conclusion/Significance

Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.     

Secretory Proteins from Adrenal Medullary Cells Are Carboxyl-Methylated In Vivo and Released Under Their Methylated Form by Acetylcholine

The carboxyl methylation of secretory proteins in vivo was investigated in bovine adrenal medullary cells in culture. Chromogranin A, the major intragranular secretory protein in adrenal medullary cells, and other secretory proteins were found to be carboxyl-methylated within secretory vesicles. The in vivo labeling pattern using [methyl-3H]methionine and the in vitro labeling pattern using S-adenosyl-[methyl-14C]methionine of intravesicular secretory proteins were similar. The detection of methylated chromogranin A in mature secretory vesicles required 3-6 h, a time consistent with the synthesis and storage of secretory proteins in this tissue. Carboxyl-methylated chromogranin A was secreted from medullary cells by exocytosis via activation of nicotinic cholinergic receptor and recovered still under the methylated form in the incubation medium. Since protein-carboxyl-methylase is cytosolic, these results suggest that methylation of secretory proteins is a cotranslational phenomenon.