A Wearable Device for Hand Sensorimotor Rehabilitation Through Augmented Sensory Feedback.

The loss of sensitivity of the upper limb due to central or peripheral neurological injuries severely limits the ability to manipulate objects, hindering personal independence. Non-invasive augmented sensory feedback techniques are used to promote neural plasticity hence to restore the grasping function. We devised a wearable device for hand sensorimotor rehabilitation capable of reliably detect transient tactile events based on custom piezoelectric polyvinylidene fluoride (PVDF) sensors and deliver discrete bursts of vibrations upon these events. We integrated the sensors into a fabric glove and tested the device in a pilot bench test exploring its ability to detect object contact and release as well as object slippage. Due to their broad bandwidth, the sensors proved to be suitable for both the applications: they responded with clear peaks when touching or releasing the object and increased the high-frequency content of the signal during slippage.

Slow Acceptance of Universal Antiretroviral Therapy (ART) Among Mothers Enrolled in IMPAACT PROMISE Studies Across the Globe.

The PROMISE trial enrolled asymptomatic HIV-infected pregnant and postpartum women not eligible for antiretroviral treatment (ART) per local guidelines and randomly assigned proven antiretroviral strategies to assess relative efficacy for perinatal prevention plus maternal/infant safety and maternal health. The START study subsequently demonstrated clear benefit in initiating ART regardless of CD4 count. Active PROMISE participants were informed of results and women not receiving ART were strongly recommended to immediately initiate treatment to optimize their own health. We recorded their decision and the primary reason given for accepting or rejecting the universal ART offer after receiving the START information. One-third of participants did not initiate ART after the initial session, wanting more time to consider. Six sessions were required to attain 95% uptake. The slow uptake of universal ART highlights the need to prepare individuals and sensitize communities regarding the personal and population benefits of the "Treat All" strategy.

Identification of a 2-propanol analogue modulating the non-enzymatic function of indoleamine 2,3-dioxygenase 1.

Indoleamine 2,3 dioxygenase 1 (IDO1) is a metabolic enzyme that catalyzes the conversion of the essential amino acid tryptophan (Trp) into a series of immunoactive catabolites, collectively known as kynurenines. Through the depletion of Trp and the generation of kynurenines, IDO1 represents a key regulator of the immune responses involved in physiologic homeostasis as well as in neoplastic and autoimmune pathologies. The IDO1 enzyme has been described as an important immune checkpoint to be targeted by catalytic inhibitors in the treatment of cancer. In contrast, a defective expression/activity of the enzyme has been demonstrated in autoimmune diseases. Beside its catalytic activity, the IDO1 protein is endowed with an additional function associated with the presence of two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which, once phosphorylated, bind SHP phosphatases and mediate a long-term immunoregulatory activity of IDO1. Herein, we report the screening of a focused library of molecules bearing a propanol core by a protocol combining microscale thermophoresis (MST) analysis and a cellular assay. As a result, the combined screening identified a 2-propanolol analogue, VIS351, as the first potent activator of the ITIM-mediated function of the IDO1 enzyme. VIS351 displayed a good dissociation constant (Kd = 1.90 µM) for IDO1 and a moderate cellular inhibitor activity (IC50 = 11.463 µM), although it did not show any catalytic inhibition of the recombinant IDO1 enzyme. Because we previously demonstrated that the enzymatic and non-enzymatic (i.e., ITIM-mediated) functions of IDO1 reside in different conformations of the protein, we hypothesized that in the cellular system VIS351 may shift the dynamic conformational balance towards the ITIM-favoring folding of IDO1, resulting in the activation of the signaling rather than catalytic activity of IDO1. We demonstrated that VIS351 activated the ITIM-mediated signaling of IDO1 also in mouse plasmacytoid dendritic cells, conferring those cells an immunosuppressive phenotype detectable in vivo. Thus the manuscript describes for the first time a small molecule as a positive modulator of IDO1 signaling function, paving the basis for an innovative approach to develop first-in-class drugs acting on the IDO1 target.

The expression of LEC/CCL16, a powerful inflammatory chemokine, is upregulated in ulcerative colitis.

Ulcerative colitis (UC) is a chronic inflammatory disease of unknown aetiology and pathogenesis. The presence in the colonic mucosa of reactive cells expressing proinflammatory cytokines and chemokines is associated with high levels of IL-10, an anti-inflammatory cytokine. Our aim was to investigate the role of IL-10 and the beta chemokine LEC/CCL16 selectively up-regulated by IL-10 in inflammatory cell recruitment and cytokine and chemokine production during UC. We studied histologically, immunohistochemically and ultrastructurally colonic biopsies from 20 active UC patients and 10 control specimens taken far from any macroscopically detectable lesion in age and sex-matched patients with noninflammatory bowel disease. In active UC, immature dendritic cells (DCs) in the LP are associated with IL-10 in the T cell rich area. Furthermore, most of the LP-infiltrating macrophages strongly expressed LEC/CCL16, a chemokine upregulated by IL-10. To evaluate if LEC/CCL16 plays a role in the inflammatory reaction present in UC, we performed morphological studies in mice injected s.c. with syngeneic tumor cells engineered to produce LEC/CCL16. We found that the LEC protein locally released by LEC-gene-transfected tumor cells is a potent proinflammatory chemokine that induces the recruitment of a reactive infiltrate, and an angiogenic process mirroring that in human UC.

Salivary carcinoma in HER-2/neu transgenic male mice: an angiogenic switch is not required for tumor onset and progression.

Morphologic examinations of salivary gland neoplasias arising in male BALB/c (H-2d) mice carrying the activated HER-2/neu (BALB-NeuT) indicate that expression of the oncogene product in the ductal-acinar structures results in a very human-like acinic cell adenocarcinoma with a smoldering course and infrequent metastatization. Typical and then atypical hyperplasia of ducts and acini preceded the rise of salivary tumors that originated from the confluence of multiple ductal hyperplastic foci, while hyperplastic acini behaved as an abortive preneoplastic lesion. The vascular network in normal, hyperplastic and neoplastic salivary tissue was analysed to see whether activation of the angiogenic process is essential in salivary gland carcinogenesis. Immunostaining with anti-endothelial cells (anti-CD31), anti-beta3 integrin and anti-laminin antibodies revealed that microvessel density was significantly higher in normal and hyperplastic than in neoplastic tissue, in which no signs of new vessel sprouting were found. Assessment of angiogenic factor expression indicates a low presence of VEGF in normal, hyperplastic and neoplastic epithelium, while bFGF was preferentially produced but not exported by neoplastic cells and remained in a cell-associated form. Our data suggest that normal salivary gland vascularization is able to support tumor onset and development with no need for an angiogenic switch.

Home collection for frequent HIV testing: acceptability of oral fluids, dried blood spots and telephone results. HIV Early Detection Study Group.

OBJECTIVE: To assess the feasibility and acceptability of bimonthly home oral fluid (OF) and dried blood spot (DBS) collection for HIV testing among high-risk individuals. DESIGN: A total of 241 participants [including men who have sex with men (MSM), injecting drug users (IDU), and women at heterosexual risk] were recruited from a randomly selected subset of study participants enrolled at four sites in the HIV Network for Prevention Trials (HIVNET) cohort, and assigned at random to bimonthly home collection of OF or DBS specimens over a 6 month interval. Participants could select telephone calls or clinic visits to receive HIV test results. METHODS: Bimonthly specimens were tracked for adherence to the schedule, were evaluated for adequacy for testing, and tested using antibody assays and polymerase chain reaction (PCR) for DBS. The acceptability of bimonthly home OF and DBS collection and telephone counseling was assessed in an end-of-study questionnaire. RESULTS: The laboratory received 96 and 90% of expected OF and DBS specimens, respectively; 99% of each specimen type was adequate for testing. Almost all (95%) participants chose results disclosure by telephone. The majority of participants (85%) reported that bimonthly testing did not make them worry more about HIV, and almost all (98%) judged that with bimonthly testing their risk behavior remained the same (77%) or became less risky (21%). CONCLUSION: Bimonthly home specimen collection of both OF and DBS with telephone counseling is acceptable and feasible among study participants at high risk. These methods will be useful for the early detection of HIV infection and remote follow-up of research cohort participants in HIV vaccine and prevention trials.

Type I consensus IFN (IFN-con1) gene transfer into KSHV/HHV-8-infected BCBL-1 cells causes inhibition of viral lytic cycle activation via induction of apoptosis and abrogates tumorigenicity in sCID mice.

In this study, we investigated the effects of human type I consensus interferon (IFN-con1) (Amgen) gene transfer into body cavity-based lymphomas (BCBL)-1 cells, which are latently infected with Kaposi's sarcoma-associated herpesvirus (KSHV) human herpesvirus-8 (HHV-8). Both the basal and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-stimulated production of KSHV/HHV-8 mature virions was strongly inhibited in genetically modified IFN-producing BCBL-1 cells as compared with parental or control transduced counterparts. A similar inhibition was obtained on treatment of parental BCBL-1 cells with exogenous IFN-con1. The reduction in KSHV/HHV-8 production was associated with a decrease in the basal and TPA-stimulated intracellular amount of the linear form of the viral genome. Interestingly, 25%40% of the IFN-producing BCBL-1 cell population underwent spontaneous apoptosis in vitro. TPA treatment, which did not significantly affect the viability of the parental and control BCBL-1 cells, resulted in the apoptotic death of up to 70% of the IFN-producing cell population. Addition of exogenous IFN-con1 to parental BCBL-1 cells produced similar effects, although less intense. Injection of either parental or control-transduced BCBL-1 cells into SCID mice resulted in progressively growing tumors characterized by an unusually high level of tumor angiogenesis. In contrast, complete tumor regression was observed in all the mice injected either subcutaneously (s.c.) or intraperitoneally (i.p.) with the IFN-producing BCBL-1 cells. These results represent the first evidence that type I IFN can counteract the activation of a productive herpesvirus infection in latently infected tumor cells by the induction of apoptosis, providing an interesting link between the antiviral and antitumor activities of this cytokine. These data suggest the possible advantages of strategies of type I IFN gene transfer (with respect to the use of the exogenous cytokine) for the treatment of patients with some HHV-8-induced malignancies.

Safety and toxicity of nonoxynol-9 gel as a rectal microbicide.

BACKGROUND AND OBJECTIVES: Methods of HIV and STD prevention, which can be controlled by the receptive partner, are a high priority for research and development. Studies on the safety of Nonoxynol-9 (N-9) on the vaginal mucosa have yielded conflicting results. No Phase I study has evaluated the effect of N-9 on the rectal mucosa. GOALS: To assess the safety of 52.5 mg of N-9 in a 1.5-g gel when applied one to four applicators per day to the rectum and penis. STUDY DESIGN: The study included 25 HIV-negative and 10 HIV-positive, monogamous gay male couples in which each partner was exclusively insertive or receptive while using N-9 gel. Each participant served as his own control during placebo gel use compared to during N-9 gel use. Receptive partners underwent anoscopic examination after 1 week of placebo use and after 2, 5, and 6 weeks of N-9 gel use, with rectal biopsies obtained after 1 week of placebo use and after 5 and 6 weeks of N-9 gel use. Insertive partners had safety monitoring after 1 week of placebo use and after 2, 5, and 6 weeks of N-9. RESULTS: No rectal ulcers were detected; superficial rectal erosions were noted in two HIV-negative participants. Abnormal or slightly abnormal histologic abnormalities of rectal biopsies were detected in 31 (89%) receptive participants after N-9 gel use compared to 24 (69%) participants after 1 week of placebo gel use. Meatal ulceration, not caused by herpes simplex virus, was detected in one HIV-negative participant. CONCLUSION: Low-dose N-9 gel was not associated with macroscopic rectal and penile epithelial disruption or inflammation, but histologic abnormalities were commonly observed during N-9 gel as well as during placebo gel use.

Acceptability of a bioadhesive nonoxynol-9 gel delivered by an applicator as a rectal microbicide.

BACKGROUND AND OBJECTIVES: Potential rectal microbicides, as an adjunct to condoms for HIV/STD prevention, have not been studied previously. GOAL OF THIS STUDY: Advantage 24 (1.5 ml of a bioadhesive gel containing 52.5 mg nonoxynol-9 administered by single-use applicator)-under investigation as a vaginal microbicide-was evaluated for acceptability among male couples. STUDY DESIGN: Twenty-five HIV-negative and 10 HIV-positive male couples participated in a frequency use escalation trial. Diaries and self-administered questionnaires assessed product use, acceptability, sexual behavior, and gastrointestinal and urologic side effects. RESULTS: Excluding participants who felt no need for an HIV prevention method, 58% said they would use Advantage 24 if approved for rectal use; 69% of receptive users reported rectal fullness and related side effects after insertion of the gel, and 68% reported applicator-related discomfort; 59% of insertive participants found the gel too sticky. CONCLUSIONS: Acceptability remains inconclusive and warrants further study of redesigned applicators and ways to minimize rectal side effects.

A "stealth effect": adenocarcinoma cells engineered to express TRAIL elude tumor-specific and allogeneic T cell reactions.

BALB/c mammary adenocarcinoma cells engineered to express TNF-related apoptosis-inducing ligand (TRAIL)/APO-2 ligand (APO-2L) on their membrane (TSA-TRAIL) grow with kinetics similar to that of parental cells (TSA-pc) in vitro and in nu/nu mice. In contrast, TSA-TRAIL cells grow faster than TSA-pc in normal BALB/c mice. In DBA/2 mice, which differ from BALB/c mice at minor histocompatibility Ags, they also grow faster and display a higher percentage of tumor takes than TSA-pc. In fully histoincompatible C57BL/6 (B6) mice, TSA-TRAIL cells form evident tumors that are slowly rejected by most mice, but outgrow in a few. In contrast, TSA-pc cells are rejected at once by B6 mice. Since TRAIL/APO-2L induces apoptosis by interacting with a variety of specific receptors, this rapid growth in both syngeneic and allogeneic mice may be the result of an immunosuppressive mechanism. The following evidence supports this hypothesis: 1) TSA-TRAIL cells overcome the strong immunity against TSA-pc cells elicited in BALB/c mice by preimmunization with TSA cells engineered to release IL-4; 2) their rejection by B6 mice does not prime a CTL-mediated memory; 3) thymidine uptake by T lymphocytes unstimulated or stimulated by allogeneic cells is inhibited when TSA-TRAIL cells are added as third party cells; 4) CTL kill TSA-pc but not TSA-TRAIL cells in 48-h assays; and 5) activated lymphocytes interacting with TSA-TRAIL cells in vivo and in vitro undergo apoptosis.

Modulation of integrin expression on mesotheliomas: the role of different histotypes in invasiveness.

The in vitro and in vivo integrin expression in human pleural malignant mesothelioma (MM) of three different histotypes was studied. Cell lines from MM of epithelioid (E1), fibrous (F1), byphasic histotype (B1) and normal mesothelial cells (NM) were analysed for the surface expression of alpha2, alpha3, alpha4, alpha5, alpha6, alphav, beta1, beta3, beta4 subunits and alphavbeta5 integrins. We found that alpha6, beta4 subunits and alphavbeta5, weakly detectable on NM cells, were expressed on MM cells. The beta3 subunit, well expressed on NM cells, was absent on MM cells. Differential expression among histotypes was observed, the MM-E1 was the least and the MM-B1 the most positive. Specimens for each MM histotype, were analysed by immunohistochemistry. The alpha6 and alphav subunits were more evident on the epithelioid histotype. Intense staining for beta3 and beta4 subunits, was found in all MM, particularly in invading cells, while the alpha5, and alphavbeta5 integrins were variously expressed. The different histotypes can affect the in vitro integrin expression and may indicate a preferential involvement of some subunits in vivo during MM tumor progression.

Migration of mesothelioma cells correlates with histotype-specific synthesis of extracellular matrix.

Three human pleural malignant mesothelioma cell cultures (MM) of epithelioid (E1), fibrous (F1) and byphasic (B1) histotype were studied in their synthesis of the extracellular matrix (ECM) components laminin (LM), fibronectin (FN), type IV collagen (cIV), and in their chemotactic and haptotactic migration towards the ECM produced proteins. MM-B1 showed the highest FN synthesis and release; MM-E1 produced the highest quantity of basement membrane constituents LM and cIV; MM-F1 weakly produced and released FN, LM and cIV. MM-B1 had the highest chemotactic and haptotactic motility, MM-F1 migrated toward the lowest concentration of LM while had reduced chemotactic activity toward FN and cIV; MM-E1 had the lowest migratory activity toward each ECM substrate. We demonstrated that three MM of different histotype are characterized by different ECM production and that these differences determine a variable ability of each MM to spread and migrate towards ECM substrates.

Treatment strategies for management of serum lipids: lessons learned from lipid metabolism, recent clinical trials, and experience with the HMG CoA reductase inhibitors.

The Adult Treatment Panel (ATP) guidelines, published initially in 1988 and revised in 1993, are based on sentinel observations and early clinical trials in support of treating and preventing coronary artery disease by cholesterol lowering. With the conclusion of several large long-term trials using HMG CoA reductase inhibitors for primary and secondary coronary prevention, the ATP II recommendations, which remain remarkably accurate, can be supplemented with more evidence-based strategies. Increasing evidence suggests that thoughtful lipid management for coronary prevention should include a more complete assessment of lipoproteins with an emphasis on apolipoproteins, triglycerides, and very low-density (VLDL) remnant particles, LDL particle size, and lipoprotein(a). This review summarizes clinically relevant lipid metabolism with an emphasis on the concept of atherogenic plasma lipids, discusses the clinical benefits and specific uses of each of the lipid-lowering drug classes, and provides an analysis of recent cholesterol-lowering primary and secondary coronary prevention trials from which a new treatment strategy can be derived.

Local release of interleukin-10 by transfected mouse adenocarcinoma cells exhibits pro- and anti-inflammatory activity and results in a delayed tumor rejection.

Tumor cells engineered to release cytokines are a valuable tool for investigating biological activities elicited by local cytokines. The parental cells of a mouse mammary adenocarcinoma (TSA-pc) were transduced with the cDNA coding for mouse interleukin-10 (IL-10). In vitro, transduced TSA cells secrete about 200 ng of IL-10/10(5) seeded cells in 48 hours (TSA-IL-10). When injected subcutaneously into syngeneic BALB/c mice, TSA-IL-10 cells gave rise to a tumor that grew progressively during the first 7-10 days and then rapidly and completely regressed. To study the events associated with this growth and disappearance, histological, immunohistochemical and ultrastructural analyses of the tumor area were performed at progressive times after challenge. A slow, but progressive and massive recruitment of leukocytes (mainly macrophages and neutrophils) into the tumor was evident. Several CD8+, CD4+ lymphocytes and a few NK cells were present. Marked inhibition of neoangiogenesis was also observed. On day 9, the microvascular network in the growth area had almost vanished, while vascular damage was present in the surrounding stromal tissue. From day 4, down-modulation of VEGF expression in the tumor area and inhibition of tumor necrosis factor-alpha (TNF-alpha) and IL-6 production by reactive leukocytes were evident. The few vessels present in the tumor area displayed poor expression of monocyte chemotactic protein-1 (MCP-1), moderate expression of VCAM-1, and strong expression of ELAM-1, three molecules that result in adhesion of inflammatory cells to the endothelium. A few tumor-infiltrating macrophages were moderately stained with anti-iNOS antibodies. These findings suggest that the collapse of established TSA-IL-10 tumors is the result of the pro- and anti-inflammatory activity of IL-10, which: a) is a signal for the local recruitment of leukocytes; b) leads to vascular damage; c) suppresses cytokine production. The coexistence of both a direct stimulatory activity on endothelial cells and an anti-angiogenic activity is evidence of the ambivalence of the local effects of IL-10.

Interaction between endothelial cells and the secreted cytokine drives the fate of an IL4- or an IL5-transduced tumour.

Injection of interleukin-4 (IL4) gene-transduced tumour cells into syngeneic immunocompetent mice resulted in tumour rejection in which a key role for eosinophils was suggested. To evaluate whether IL5 inhibits tumour growth by selectively inducing eosinophil recruitment and activation, a poorly differentiated mammary adenocarcinoma cell line (TSA) was transfected with the IL5 gene and the cells secreting IL5 (TSA-IL5) were injected subcutaneously (s.c.) in syngeneic mice. The oncogenicity of TSA-IL5 was compared with that exhibited by TSA cells transfected with the IL4 gene (TSA-IL4) and with the neomycin resistance gene only (TSA-neo). At progressive times after subcutaneous challenge, tumour growth areas were studied histologically, ultrastructurally, and immunohistochemically to identify the reactive cells, visualize tumour vessels, and detect the cytokines and chemokines involved in the anti-tumour reaction. Both the morphological and the functional data showed that TSA-IL5, despite the large eosinophil infiltrate, grew progressively like TSA-neo, suggesting that eosinophils per se do not play a crucial role in TSA tumour rejection. Furthermore, our data indicate that the rejection of TSA-IL4 depends on the IL4-induced expression of VCAM-1 and MCP-1 by endothelial cells. MCP-1 together with VCAM-1 results in recruitment and activation of basophils, mast cells, and macrophages, and hence a pro-inflammatory cytokine cascade that initially favours the influx and activation of neutrophils and finally tumour rejection. In this context, the rejection of TSA-IL4 seems to involve a variety of reactive cells and rests on a continuous cross-talk between basophils, mast cells, macrophages, CD8-positive lymphocytes, and granulocyte subsets, mostly neutrophils.

Intraoperative dosimetry of 90Y in liver tissue.

The use of an intraoperative radiation detection probe was assessed following the injection of 90Y labelled radioactive microspheres into the normal liver of 10 sheep. The absorbed dose to the liver ranged from 10 to 50 Gy. There was a linear relationship between the probe recordings and the radiation dose as measured by scintillation counting of liver tissue. By using the probe during the procedures for internal radiation therapy the injected dose of radioactive microspheres can be manipulated to provide the maximum tolerable dose to normal tissue.

Effect of angiotensin II on blood flow in the transplanted sheep squamous cell carcinoma.

The effects of systemic infusion of angiotensin II on the distribution of blood flow in the sheep squamous cell carcinoma after transplantation to the liver were measured using tracer microspheres. The ratio of arterially introduced radioactive microspheres embolizing in tumour tissue compared to normal hepatic parenchyma was measured before and after infusion of angiotensin II. Doses of angiotensin II inducing increases in mean arterial blood pressure of 26 mmHg produced significant increases in the embolization ratio from 2.8 to 4.1:1. In addition, the ratio of microspheres gaining access to the necrotic centres of the tumours compared to the normal liver tissue significantly increased from 1.6 to 2.3:1. In terms of the technique of internal radiation therapy for hepatic metastases, the concurrent infusion of angiotensin II with injection of radioactive microspheres would result in a substantially enhanced radiation dose to liver tumours. At the same time the dose to normal tissue can be minimized.

The importance of quantitative electron microscopy in studying hypertrophic neuropathies. A comparison between a case of Déjérine Sottas disease (HMSN III) and a case of the hypertrophic form of Charcot-Marie-Tooth disease (HMSN I).

The electron microscopic features and quantitative morphometric data of the sural nerve in Déjérine-Sottas disease (HMSN III) and in the hypertrophic form of Charcot-Marie-Tooth disease (HMSN I) are reported. Both forms are characterized by onion bulb formations, but they differ in: a) increased incidence of mucoid connective tissue in DS disease; b) higher incidence of demyelination in DS disease; c) uniformly small size of the remaining myelinated fibers in DS disease; d) larger and more developed onion bulbs in DS disease; e) clusters of 2-4 myelinated fibers in the core of onion bulbs of CMT disease; f) peripheral concentric lamellae with the typical aspects of the denervation bands of the unmyelinated fiber type in the onion bulbs of the CMT disease; g) non-involvement of unmyelinated fibers in DS disease. These differences permit two types of onion bulbs to be distinguished, and suggest a different pathogenesis. Electron microscopy, coupled with quantitative determinations, permits a deeper insight into the formation modalities of onion bulbs and may help in the diagnosis of the different forms of hypertrophic neuropathies.

Apraxia in deep cerebral lesions.

In a series of 50 patients with cerebrovascular lesions (demonstrated with CT scan), seven patients had lesions located in the basal ganglia and/or thalamus. All these seven patients were apractic. Ideomotor apraxia was present in all patients; five also had constructional apraxia, and one had bucco-facial apraxia. None of the patients had utilisation apraxia. These observations indicated that apraxia is not only a "high cerebral (cortical) function", but may depend also on the integrity of subcortical circuits and structures.

Selective release of glutamate from cerebellar granule cells differentiating in culture.

The aim of the present study was to assess whether endogenous and newly synthesized glutamate can be released from differentiating cultured cerebellar granule cells in a way compatible with a neurotransmitter role. Granule cells from 8-day-old rat cerebella were grown in basal Eagle's medium with 10% fetal calf serum for 2-12 days in vitro (DIV), then washed with Krebs-Ringer medium, and labeled for 45 min with tracer amounts of radioactive glutamine. Subsequently, the release of endogenous glutamate and of newly formed radioactive glutamate was measured in basal conditions and upon depolarization with elevated K(+) concentration or veratridine. At 2 DIV, the release of endogenous and newly synthesized glutamate evoked by high K(+) concentration was small and Ca(2+) independent, but it progressively and steadily increased (up to 8- to 10-fold) and became Ca(2+) dependent (up to 80-85%) at later stages (4, 8, and 12 DIV). Veratridine was almost ineffective with cells at 2 DIV but greatly increased glutamate release (endogenous and neosynthesized) at 8 DIV, and its action was totally antagonized by tetrodotoxin. The level and synthesis of glutamate remained fairly constant in cells from 2 to 12 DIV. gamma-Aminobutyric acid synthesis from radioactive glutamine was about 3% of that of glutamate, and gamma-aminobutyric acid release (endogenous and neosynthesized) was not measurable. Aspartate synthesis was about 10% of that of glutamate, and the high K(+) concentration-evoked release of this amino acid was modest and scarcely affected by Ca(2+). Neither high K(+) concentration nor veratridine was able to induce glutamate release from confluent cerebellar astrocyte cultures at 14 DIV, although the level and synthesis of the amino acid were comparable to those in granule cells. In conclusion, the data show that a stimulus-coupled release of endogenous and neosynthesized glutamate is progressively expressed by cerebellar granule cells differentiating in culture, and this strongly supports the concept that glutamate is the neurotransmitter of these cells.