T cells of multiple sclerosis patients target a common environmental peptide that causes encephalitis in mice.

Multiple sclerosis (MS) is a chronic autoimmune disease triggered by unknown environmental factors in genetically susceptible hosts. MS risk was linked to high rates of cow milk protein (CMP) consumption, reminiscent of a similar association in autoimmune diabetes. A recent rodent study showed that immune responses to the CMP, butyrophilin, can lead to encephalitis through antigenic mimicry with myelin oligodendrocyte glycoprotein. In this study, we show abnormal T cell immunity to several other CMPs in MS patients comparable to that in diabetics. Limited epitope mapping with the milk protein BSA identified one specific epitope, BSA(193), which was targeted by most MS but not diabetes patients. BSA(193) was encephalitogenic in SJL/J mice subjected to a standard protocol for the induction of experimental autoimmune encephalitis. These data extend the possible, immunological basis for the association of MS risk, CMP, and CNS autoimmunity. To pinpoint the same peptide, BSA(193), in encephalitis-prone humans and rodents may imply a common endogenous ligand, targeted through antigenic mimicry.

Peptide dose, MHC affinity, and target self-antigen expression are critical for effective immunotherapy of nonobese diabetic mouse prediabetes.

Cross-reactive T cells that recognize both Tep69 (dominant nonobese diabetic (NOD) T cell epitope in ICA69 (islet cell autoantigen of 69 kDa)) and ABBOS (dominant NOD T cell epitope in BSA) are routinely generated during human and NOD mouse prediabetes. Here we analyzed how systemic administration of these mimicry peptides affects progressive autoimmunity in adoptively transferred and cyclophosphamide-accelerated NOD mouse diabetes. These models were chosen to approximate mid to late stage prediabetes, the typical status of probands in human intervention trials. Unexpectedly, high dose (100 microg) i.v. ABBOS prevented, while Tep69 exacerbated, disease in both study models. Peptide effects required cognate recognition of endogenous self-Ag, because both treatments were ineffective in ICA69null NOD congenic mice adoptively transferred with wild-type, diabetic splenocytes. The affinity of ABBOS for NOD I-A(g7) was orders of magnitude higher than that of Tep69. This explained 1) the expansion of the mimicry T cell pool following i.v. Tep69, 2) the long-term unresponsiveness of these cells after i.v. ABBOS, and 3) precipitation of the disease after low dose i.v. ABBOS. Disease precipitation and prevention in mid to late stage prediabetes are thus governed by affinity profiles and doses of therapeutic peptides. ABBOS or ABBOS analogues with even higher MHC affinity may be candidates for experimental intervention strategies in human prediabetes, but the dose translation from NOD mice to humans requires caution.

Persistent T cell anergy in human type 1 diabetes.

An anergic phenotype has been observed in nonobese diabetic (NOD) mice and some autoreactive T cells from patients with type I diabetes. To better understand this phenomenon, we measured T cell proliferative responses to 10 diabetes-associated and up to 9 control Ags/peptides in 148 new diabetic children, 51 age- and MHC (DQ)-matched siblings (sibs), 31 patients with longstanding diabetes, and 40 healthy controls. Most (78-91%) patient and sib responses to glutamate decarboxylase of 65 kDa (GAD65), islet cell cytoplasmic autoantibody (ICA) 69, diabetes-associated T cell epitopes in ICA69 (Tep69), and heat shock protein (Hsp) 60 involved anergic T cells that required exogenous IL-2 to proliferate. Responses to proinsulin, IA-2 (and tetanus toxoid) required no IL-2 and generated sufficient cytokine to rescue anergic T cell responses. Most new patients (85%) had autoreactive T cells, three quarters targeting more than half of the diabetes Ags. Only 7.8% of the sibs and none of the controls had such multiple T cell autoreactivities, which thus characterize overt disease. Multiple anergic and nonanergic T cell autoreactivities were sustained during 2 yr follow-up after onset and in patients with longstanding (3-26 yr) diabetes. Activated patient T cells survived severe IL-2 deprivation, requiring 20-100 times less IL-2 than normal T cells to escape apoptosis. Diabetic T cell anergy thus persists for decades and is Ag and host specific but not related to disease course. Rescue by IL-2 from bystander T cells and high resistance to apoptosis may contribute to this persistence. These data explain some of the difficulties in the routine detection of disease-associated T cells, and they emphasize challenges for immunotherapy and islet transplantation.

Nasopharyngeal brush biopsies and detection of nasopharyngeal cancer in a high-risk population.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an important tumor in many countries. Ethnic and regional factors strongly influence disease risk. NPC is usually diagnosed late in disease development, and 10-year survival rates are as low as 10%. Epstein-Barr virus (EBV), a possibly causative agent, is present in all cells of essentially all undifferentiated NPCs. We wished to determine the following: 1) whether an ambulatory nasopharyngeal brush biopsy could provide sufficient tumor cell DNA for the detection of EBV and 2) whether the detection of EBV in this locale reflects the presence of tumor cells or simply EBV carrier status. METHODS: We collected nasopharyngeal tissue via ambulatory brush biopsies from 21 patients with newly diagnosed NPC and from 157 subjects with other otolaryngologic complaints. The majority of study subjects were from high-risk populations. Sample DNA was analyzed for the presence of EBV genomic sequences by use of the polymerase chain reaction (PCR). RESULTS: Ninety-six percent of samples yielded sufficient DNA for PCR amplification. Nineteen of 21 patients with NPC brushed positive for EBV DNA, while all but two (1.3%) of 149 informative control subjects were negative for EBV (two-sided P<.0001). One of the EBV-positive control subjects had an EBV-positive inverted sinonasal papilloma; the other EBV-positive control subject exhibited no overt clinical disease. CONCLUSION: Demonstration of EBV DNA in nasopharyngeal brush biopsy specimens detects NPC with a sensitivity of at least 90% (95% confidence interval = 89.63%-91.32%) and a specificity of approximately 99% (95% confidence interval = 98.64%-98.68%). This technique merits further testing as a possible ambulatory screening strategy in high-risk populations.

The physiologic reservoir of Epstein-Barr virus does not map to upper aerodigestive tissues.

The human Epstein-Barr herpesvirus (EBV) has distinct oncogenic potential, but with over 90% of the adult world population infected, malignancy is a rare outcome of carrier status. However, EBV's association with over half of Hodgkin's and non-Hodgkin's lymphomas as well as several solid tumors, notably nasopharyngeal carcinoma, makes EBV-linked malignancies one of the largest single cancer entities. EBV is a B-lymphotropic virus, well controlled by surveillant T cells in immunocompetent hosts. To determine the presence and site of principal virus reservoirs is a likely prerequisite for understanding the etiology of EBV-associated tumors. Its near 100% association with nasopharyngeal carcinoma led to postulates that the upper aerodigestive tract tissue may be common sites of persistent latent or low-grade replicating infection. Using a protocol designed to avoid viral crosscontamination, the authors employed polymerase chain reaction to detect genomic EBV DNA sequences in 231 biopsies from different mucosal sites in the upper aerodigestive tract, as well as from salivary gland tissue and neck nodes in individuals not suspected to have EBV-related malignancy. Only two samples, one from oral cavity mucosa and one from parotid gland tissue, were positive for EBV. The observation that oropharyngeal tissue is not the principal EBV reservoir has mechanistic implications for the development of EBV-positive tumors in that locale.

Immunological aspects of nutritional diabetes prevention in NOD mice: a pilot study for the cow's milk-based IDDM prevention trial.

Human epidemiological studies delineated early exposure to intact dietary protein (e.g., most infant formulas) as an environmental risk factor for the development of IDDM. The Trial to Reduce IDDM in the Genetically at Risk (TRIGR), an international IDDM prevention trial, has been designed to determine if avoidance of intact dairy protein in high-risk infants < or =6 months of age can reduce the subsequent diabetes incidence. We here studied the casein hydrolysate-based trial diet (Nutramigen) in NOD mice. When given either continuously or for 10 weeks after weaning, the test diet was highly effective in preventing autoimmune diabetes (32-week incidence: 4.6 vs. 58.8%) and in preserving pancreatic insulin levels, with little effect on islet inflammation. Spleen cells from protected NOD mice failed to adoptively transfer diabetes into irradiated syngeneic recipients. When co-transferred with splenocytes from diabetic donors, cells from diet-protected mice inhibited adoptive diabetes transfer (incidence 50 vs. 94%, P < 0.001). T-cell reactivity to the islet cell autoantigens ICA69 (islet cell antigen 69) and GAD65 developed only in diabetic recipients of spleen cell grafts, indicating that diabetes protection extends to more than one autoantigen. In protected mice, ICA69 T-cell reactivity was not detectable spontaneously nor after priming with this autoantigen; however, priming with the cross-reactive non-self-antigen bovine serum albumin recruited T-cells responsive to ICA69. Thus, diabetes prevention with the clinical trial diet is effective in NOD mice, where it affects some T-cell repertoires and allows development of regulatory cells that interfere with destructive autoimmunity.

Role of Epstein-Barr virus in fine-needle aspirates of metastatic neck nodes in the diagnosis of nasopharyngeal carcinoma.

BACKGROUND: The patient with nasopharyngeal carcinoma (NPC) frequently is initially seen with regional node dissemination. Preliminary investigations suggest that the presence of Epstein-Barr virus (EBV) genomes in neck metastases from an occult primary may be diagnostic and predictive of NPC. The goal of this study was to test this proposition. METHODS: The polymerase chain reaction (PCR) was used to detect the presence of EBV DNA in fine-needle aspirate (FNA) samples obtained from malignant neck nodes. Control samples were obtained from other locations in the head and neck. PATIENTS: The patients in this study were evaluated at the Toronto Princess Margaret Hospital, a province-wide tertiary-care cancer treatment center. Of the 23 patients evaluated with malignant neck masses, 6 had NPC, 5 patients had metastatic squamous cell carcinoma of an unknown primary, and 12 patients served as controls with other known head and neck carcinomas. One of the patients initially diagnosed as an unknown primary later demonstrated NPC. FNA specimens were also obtained from 24 normal parotid, submandibular, or thyroid glands for comparison. RESULTS: In the samples with sufficient DNA for analysis, EBV was detected in 5 of 5 neck nodes from patients with known NPC. EBV was also detected in the neck node of a patient who went on to develop NPC and in a cervical node from 1 of 2 patients in whom the primary tumor remained unknown. None of the evaluable control neck nodes of FNA controls from other sites demonstrated EBV. CONCLUSIONS: These results demonstrate the utility of NPC-diagnostic EBV gene amplification in FNA samples of neck metastases and suggest that the presence of the EBV genome in FNA samples of neck nodes is predictive of the presence of NPC.

A majority of inverted sinonasal papillomas carries Epstein-Barr virus genomes.

BACKGROUND: The relationship between sinonasal inverted papilloma (IP) and various strains of human papilloma virus (HPV) has been examined previously. Yet there is little consensus regarding the incidence or role of HPV in IP. The possible role of Epstein-Barr virus (EBV), which, like HPV, is a DNA virus linked to human lymphoid and epithelial malignancies, was investigated. METHODS: The polymerase chain reaction (PCR) was used to detect EBV genomic sequences in surgical specimens of IP, in benign nasal polyps, and various control tissues. The IP specimens were similarly examined for the presence of HPV types 6, 11, 16, and 18. RESULTS: EBV DNA was found in 13 of 20 IP specimens (65%) and none of the 10 control tissues. Nine of the 20 specimens contained HPV DNA, and 5 of 20 specimens contained both EBV and HPV. CONCLUSIONS: These results imply a previously unsuspected role for Epstein-Barr virus in the pathogenesis of sinonasal inverted papilloma.

T cell activation and anergy to islet cell antigen in type I diabetes.

Early exposure to cow milk proteins was linked to the development of type I diabetes by consistent epidemiology, and by feeding and tolerization studies in diabetes-prone rodents. Dietary BSA was suggested as the culprit because patients and relevant rodents have elevated anti-BSA Abs that precipitate the recently cloned protein, p69, from beta cell lysates. A total of 68 of 78 children with recent onset diabetes had BSA-reactive T cells at the time of diagnosis. Here we 1) map the fine specificity of these T cells, 2) delineate a homologous peptide sequence near the N-terminus of p69, and 3) demonstrate T cell recognition of this p69 sequence (T cell epitope p69, Tep69) by patient T cells. The Tep69 sequence is conserved in p69 of patients and diabetes-prone rodents. Whereas BSA triggers T cell proliferation, recombinant p69 and a synthetic Tep69 peptide induce early stages of T cell activation (IL-2R transcription) but insufficient IL-2 production and thus anergy. Exogenous IL-2 overrides anergy and allows proliferative expansion of p69-responsive T cells. In mixing experiments, p69 and Tep69 peptide prevented proliferative responses to BSA even at 100-fold smaller concentrations. These findings imply that high-affinity self-peptide triggers anergy, whereas low-affinity mimicry Ag triggers proliferative expansion of these T cells. This implies a disease model in which mimicry Ag would rescue autoreactive cells from ablation by self-Ag.

Cloning of human and rat p69 cDNA, a candidate autoimmune target in type 1 diabetes.

Triggering of autoimmunity in insulin-dependent diabetes was linked to dietary bovine serum albumin (BSA). Anti-BSA antibodies from diabetes-prone rats precipitate a protein, p69, from islet cell lysates. We have used these antibodies to identify rat p69 cDNAs. Human p69 cDNA was identified by crosshybridization. The p69 coding regions show 87% nucleotide and 89% amino acid homology. Recombinant p69 is recognized by autoantibody and T cells from diabetic children.

Epstein-Barr virus and nasopharyngeal carcinoma: bringing molecular genetics strategies to head and neck oncology.

In this article, we consider the tools of molecular genetics and strategies that have, or likely will have, an impact in otolaryngology, either as diagnostic tools or as strategies, with more far-reaching applications in tumour therapy, relapse monitoring, and ultimately, approaches to tumour prevention. Nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV). Detection of the virus following gene amplification by the polymerase chain reaction (PCR) can provide a diagnostic tumour marker, both in primary and metastatic sites. NPC can be considered as a model disease on which molecular genetics is and likely will be of considerable impact. NPC is characterized by the presence of a genetically stable, viral agent of proven oncogenicity. The presence of attractive experimental systems for the study of EBV-associated tumours and their accessibility may combined with new molecular approaches towards diagnostic and, eventually, therapeutic improvements in the treatment of this clinically ominous malignancy.

Epstein-Barr virus surveillance after renal transplantation.

At least 1% of organ transplant recipients develop Epstein-Barr virus-positive, often fatal lymphomas. EBV-positive cells accumulating in some organ transplant recipients were suggested to predict EBV+ lymphoma risk but no prospective study has been reported. We used the polymerase chain reaction (PCR) to detect EBV genomic sequences in successive blood samples of 60 kidney recipients before and up to 11 years after renal transplantation. Xenotransplantation of EBV-positive patient and -negative control samples into mice with severe combined immunodeficiency (SCID) was used to assess the tumor risk inherent in these samples. Despite single EBV+ cell detection sensitivity, none of the control samples was positive for EBV genomic sequences. In nearly 2/3 of patients EBV genomic DNA was detectable 3-6 months after transplantation for about 3 months. No patient developed lymphoma. Lymphocytes from 8 EBV-genome positive patients and 10 healthy donors were engrafted into 38 SCID mice. Human B cell lymphoma developed in 75% of the control grafts within about 3 months. In striking contrast, none of the patient grafts developed lymphoma despite the large numbers of EBV+ cells initially transplanted. Patient lymphocyte grafts were resistant to injection of live EBV, while in control lymphocyte grafts this caused lymphoma development within 3 weeks. We conclude that a 100-1000-fold expansion of circulating EBV+ B cell pools occurs frequently after organ transplantation and that it is balanced by effective EBV immunosurveillant functions resistant to immunosuppression. The mere detection of EBV genomic material was not predictive of lymphoma development.

Reversion of the SCID phenotype by human T cell grafts. Development of cross-species immunocompetence.

Due to defective recombinase function, mice with severe combined immunodeficiency (SCID) lack functional lymphocytes and can accept human lymphoid xenografts. Xenografted animals (SCIDhum) are thought to provide a neutral environment for in vivo studies of normal, malignant or HIV-infected human cells. SCIDhum often develop endogenous, EBV+ lymphomas in the graft and in the our study two-thirds of 142 SCIDhum mice did so. Surprisingly, one-third of animals developed reversion of the SCID phenotype rapidly after human T cell engraftment. 90% of tumors occurred in nonrevertant and only 10% in revertant mice. These revertant animals showed immunologic tolerance for normal human B lymphocytes, maintained stable levels of mouse and human IgM and IgG. In addition, they generated competent mouse T cells able to kill transformed (EBV+) but not fresh B cells from the same donor nor unrelated human B cell lines. The tolerance for human lymphoid cells and the cross-species antitumor competence of host T lymphocytes imply unexpected recognition and selection events. Rather than a neutral "bioreactor," these observations mark the SCID host as potentially active participant in a composite immune system generated by xenografting.

Viral interleukin 10 is critical for the induction of B cell growth transformation by Epstein-Barr virus.

We have used an efficient cDNA subtraction library procedure to identify newly induced genes in human B lymphocytes infected for 6 h with Epstein-Barr virus (EBV). Among the genes identified by automated sequencing of a random subset of clones from this library, one coded the EBV BCRF1 open reading frame, which specifies the viral interleukin 10 gene (vIL-10). This molecule is highly homologous to human (h)IL-10 and was previously thought to represent a "late" viral gene expressed only during the lytic phase of virus replication. Using gene amplification by reverse transcriptase polymerase chain reaction of B cell RNA obtained at varying times after infection, we detected vIL-10 expression within a few hours of EBV infection, followed, 20-30 h later by expression of hIL-10. Expression of both genes continued beyond the initial transformation phase (5-10 d) and was present in all transformed cell lines tested. When added at the time of viral infection, antisense (but not sense) oligonucleotides for vIL-10 mRNA (cytosolic half-life, approximately 6 h) prevented subsequent B cell transformation. The antisense effect was highly specific, leaving the expression levels of other transformation-related genes intact. Addition of exogenous (h)IL-10 rescued the transformation process in antisense-treated cells. Our observations establish vIL-10 as a new latency gene with a directly transformation-prerequisite function.

Unexpected patterns of Epstein-Barr virus gene expression during early stages of B cell transformation.

Following Epstein-Barr virus (EBV) binding to its CD21 cell surface receptor, virus internalization, nuclear translocation, and circularization of the viral episome were found to occur within 30 min, immediately preceding the expression of EB nuclear antigen (EBNA)-1 and -2 and latent membrane protein (LMP)-1 and -2 genes. Early viral gene expression was unaffected by blockade of the virus induced, transformation-prerequisite cellular activation pathway (Ca2+ currents, tyrosine phosphorylation, induction of p56lck, hsp70, and hsp90). Despite life times of only 3 h, antisense (but not sense) oligonucleotides for the above latency genes prevented subsequent transformation. Any one antisense oligonucleotide dramatically depleted transcripts not only of the target gene, but of all other latency genes. The blocking effect of antisense oligonucleotides allowed us to identify a new transformation-prerequisite latency gene near the fused termini. The concerted regulation of EBV gene expression is highly unusual and unexplained but our results imply critical, perhaps regulatory roles for initial latency gene transcripts themselves.

Detection of viral sequences by internally calibrated gene amplification.

Inherent pitfalls of the polymerase chain reaction (PCR) can become serious difficulties when transferring research applications to high-volume routine procedures such as biofermentation process control and clinical diagnostics. Difficulties include 1) the danger of accidental sample contamination with positive control templates; 2) variable amplification due to positional effects in thermocycler blocks and unequal primer efficiency for sense/anti-sense strands; and 3) the need for reliable controls, which provide confidence for reporting negative reactions. Using the PCR detection system for Epstein-Barr virus as a model, we have developed a quick process to generate mutant internal co-amplification templates. These can be used for titration of amplification sensitivity. More importantly, single tube co-amplification without titrations allows determination of the minimum sensitivity achieved in each individual reaction; critical information when reporting negative diagnostic results. Mutant and native fragments are easy to distinguish by size, and sample cross contamination can be readily identified. The system should be easily adaptable to gene amplification procedures, which aim to routinely detect the presence of a given gene fragment in a controlled fashion.

The growth transformation of human B cells involves superinduction of hsp70 and hsp90.

Epstein-Barr virus (EBV) is a latent human herpes virus associated with a range of malignant and non-malignant disorders. EBV binds to CD21 virus receptors on B lymphocytes and growth transforms these cells; in susceptible (e.g., immunodeficient) hosts such cells rapidly expand into fatal lymphomas. Virus binding and infection trigger a cascade of cellular events which are transformation prerequisite and analogous to non-oncogenic cell activation events but which differ in several quantitative or qualitative respects. Unique trans-membrane Ca2+ currents, Na+/H+ exchange, as well as tyrosine phosphorylation and p56lck-gene induction suggest that even early on the transformation process has oncogenic specificity. In this report we describe that two additional cellular gene families, the stress proteins hsp70 and hsp90, are coordinately induced at mRNA and protein levels and, quite different from hsp induction by thermal stress, this induction is dependent on EBV-induced trans-membrane Ca2+ currents. Blockade of hsp induction prevents transformation. The kinetics and induction prerequisites set this response well apart from reported responses to thermal or viral stress protein induction. Like p56lck-, hsp induction is purely a post-receptor binding event and not dependent on expression of any viral gene. The induction kinetics, with a peak at approximately 12-16 hr and subsequent decline to control levels, considerably extend the chronological map of elements in the CD21-dependent branch of the transformation pathway and suggest a specific role of induced hsp different from the cell cycle-related functions observed in other cell systems.

Exogenous but not endogenous EBV induces lymphomas in beige/nude/xid mice carrying human lymphoid xenografts.

Epstein--Barr virus (EBV) is a latent human herpes virus that growth-transforms EBV receptor/CD21+ B cells and is associated with several high-frequency malignancies. Reactivation of latent EBV occurs in approximately 1/3 of organ graft recipients and a majority of AIDS patients; EBV-positive B lymphoproliferative lesions represent often fatal complications in organ transplantation and late-stage AIDS. Although such lymphomas can arise from endogenous virus, the high tumor risk in EBV-seronegative transplant recipients implies de novo infection, in particular virus transmission with intra-graft B lymphocytes. Since SCID mice engrafted with human lymphocytes (SCIDhum) typically develop endogenous EBV+ (human) tumors in their graft it is difficult to study exogenous virus transmission in this model. We here demonstrate that beige/nude/xid mice engrafted with human lymphoid cells (BNXhum) selectively accept human B but not T cell grafts. Unexpectedly these mice fail to develop endogenous lymphomas observed in SCIDhum mice engrafted in parallel. However, injection of as few as less than 500 EBV particles produces rapidly fatal, polyclonal lymphomas in BNXhum animals. This virus sensitivity of BNXhum approaches conditions for EBV transmission with organ grafts.

The tyrosine kinase lck is critically involved in the growth transformation of human B lymphocytes.

Epstein-Barr virus (EBV) exposure of human B lymphocytes induces rapid, Ca(2+)-dependent tyrosine phosphorylation of two cytosolic proteins, one likely the CD21 EBV receptor and another unknown species of 55-60 kDa. We now identify the latter protein as the tyrosine kinase lck (p56lck). In T cells many activation events reduce the high constitutive p56lck expression levels typical for that lineage, and they induce the appearance of a 60-kDa lck species. We now demonstrate that in B cells exposed to EBV the at best low constitutive p56lck expression levels are rapidly and transiently up-regulated without generation of 60-kDa lck. lck-specific antisense oligonucleotides block p56lck induction and prevent subsequent B cell activation and immortalization whereas B cell activation by nononcogenic agents was unaffected. We propose that p56lck superinduction is a transformation prerequisite which signals entry into the oncogenic growth transformation process.

Dissociation of unidirectional influx of external Ca2+ and release from internal stores in activated human T lymphocytes.

Addition of lectin or antibody to the T cell receptor complex of human T cells results in a rapid increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This response is biphasic and results from contributions of Ca2+ from internal stores, uptake of Ca2+ across the plasma membrane and possibly a decrease in Ca2+ efflux. These responses have been linked through the activity of inositol 1,4,5-trisphosphate in releasing Ca2+ from internal stores and potentially mediating Ca2+ uptake across the plasma membrane. Following addition of phytohemagglutinin or anti-CD3 antibody to resting T cells or Jurkat cells, we have been able to dissociate the [Ca2+]i responses by loading cells with the Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA). In BAPTA-loaded T cells, we have shown that Ca2+ mobilized from intracellular stores following activation is effectively buffered, while stimulated Ca2+ uptake and associated changes in [Ca2+]i were relatively unaffected. In this report, we show that the sustained increase in [Ca2+]i is due to increased unidirectional influx of external Ca2+ without changes in efflux and that it is the entry of extracellular Ca2+ which is sensitive to the transmembrane potential.