Extracellular recordings from locally dense microelectrode arrays coupled to dissociated cortical cultures.

High-density microelectrode arrays (MEAs) enabled by recent developments of microelectronic circuits (CMOS-MEA) and providing spatial resolutions down to the cellular level open the perspective to access simultaneously local and overall neuronal network activities expressed by in vitro preparations. The short inter-electrode separation results in a gain of information on the micro-circuit neuronal dynamics and signal propagation, but requires the careful evaluation of the time resolution as well as the assessment of possible cross-talk artifacts. In this respect, we have realized and tested Pt high-density (HD)-MEAs featuring four local areas with 10microm inter-electrode spacing and providing a suitable noise level for the assessment of the high-density approach. First, simulated results show how possible artifacts (duplicated spikes) can be theoretically observed on nearby microelectrodes only for very high-shunt resistance values (e.g. R(sh)=50 kOmega generates up to 60% of false positives). This limiting condition is not compatible with typical experimental conditions (i.e. dense but not confluent cultures). Experiments performed on spontaneously active cortical neuronal networks show that spike synchronicity decreases by increasing the time resolution and analysis results show that the detected synchronous spikes on nearby electrodes are likely to be unresolved (in time) fast local propagations. Finally, functional connectivity analysis results show stronger local connections than long connections spread homogeneously over the whole network demonstrating the expected gain in detail provided by the spatial resolution.

High-resolution MEA platform for in-vitro electrogenic cell networks imaging.

A platform based on an active-pixel-sensor electrode array (APS-MEA) for high-resolution imaging of in-vitro electrogenic cell cultures is presented, characterized and validated under culture conditions. The system enables full frame acquisition at 8 kHz from 4096 microelectrodes integrated with separations of 21 microm and zoomed area acquisition with temporal resolutions down to 8 micros. This bi-modal acquisition feature opens new perspectives in particular for neuronal activity analysis and for the correlation of micro-scale and macro-scale behaviors. The low-noise performances of the integrated amplifier (11 microVRMS) combined with a hardware implementation reflecting image-/video-concepts enable high-resolution acquisitions with real-time processing capabilities adapted to the handling of the large amount of acquired data.

Decreased IL-4 production in new onset type I insulin-dependent diabetes mellitus.

IL-4 has been shown to protect against diabetes development in rodent models of insulin-dependent (type I) diabetes mellitus (IDDM). To study IL-4 production in human IDDM, PBMC from IDDM patients and controls were stimulated in vitro with PHA, anti-CD3 mAb, or PMA and ionophore. IL-4 production by PBMC or T cells was strongly impaired in IDDM patients at diabetes onset (p < 0.0001). The mean IL-4 response of patients in the honeymoon stage was higher than the mean of the new onset patients, but significantly lower than the control group (p = 0.01). Patients with IDDM of longer duration (>2 yr) showed a wide range of IL-4 responses and their mean IL-4 response was lower than the controls; however, the difference was not statistically significant. IL-4 mRNA levels were measured using competitive reverse transcription PCR. The results showed greatly reduced mRNA levels in new onset IDDM. In contrast, IL-1 production (measured by ELISA) and IFN-gamma mRNA (measured by reverse transcription PCR) were not significantly different in IDDM. The results suggest an imbalance of inflammatory vs anti-inflammatory cytokine production at the onset of IDDM. Deficient IL-4 production as seen at the onset of IDDM may play a role in the development of diabetes by allowing the inflammatory/autoimmune process in pancreatic islets to progress.

IL-4 expression in human T cells is selectively inhibited by IL-1 alpha and IL-1 beta.

Imbalances in anti-inflammatory and proinflammatory cytokines may be responsible for initiation or progression of diverse pathologic states including autoimmune and infectious diseases. IL-4 production of proinflammatory cytokines and IL-12 promotes differentiation and activation of IFN-gamma-producing T cells, but does a counter-regulatory effect of proinflammatory cytokines on IL-4 production exist? This study evaluates the effect of proinflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-12, and TNF-alpha) on IL-4 production in primary human T cell cultures. PBMCs from healthy individuals were tested for IL-4 production in response to PHA and various cytokines. IL-4 was measured by proliferation of the IL-4-sensitive T cell line (CT.h4S) or ELISA. IL-1 alpha and IL-1 beta inhibited IL-4 production by 20 to 80% in > 92% of healthy individuals (p = 0.0001, paired t-test). IL-12 had an inhibitory effect on PBMC IL-4 production as previously described, but neither IL-6 nor TNF-alpha inhibited IL-4 production. IL-1 had no effect on PHA-induced PBMC or purified T cell proliferation or IL-2 production. IL-4 production by purified T cells stimulated by PHA or the combination of PMA with calcium ionophore (A23187) was inhibited by IL-1, and reconstitution with peripheral blood-derived adherent macrophages had no effect. IL-12 did not inhibit IL-4 production in stimulated purified T cells. Steady state IL-4 mRNA levels were determined by semiquantitative competitive reverse transcribed PCR (RT-PCR). Marked inhibition of IL-4 mRNA levels were seen at 5 h after exposure to IL-1. This interaction between IL-1 and IL-4 may be an important physiologic regulator of the balance between proinflammatory cytokines from activated macrophages and anti-inflammatory cytokines from T cells.

HIV type 1 induction of interleukin 1 and 6 production by human thymic cells.

In vitro and in vivo studies have demonstrated that HIV can infect thymocytes at different maturational stages and lead to changes in the thymic microenvironment. To determine the effect of HIV on thymic stromal cells and the production of cytokines important in thymocyte development, three types of adherent thymic cultures were established and studied: thymic epithelial cells (TECs), macrophage-enriched, and mixed cultures of macrophages and TECs (M phi/TEC). Cultures were exposed to HIV-1 strains HIV-1IIIB and HIV-1Ba-L, and studied from day 2 to day 26 for the presence of infection, cytopathology, and cytokine (IL-1 alpha, IL-1 beta, and IL-6) production. M phi/TEC and macrophage-enriched cultures were infected by both HIV strains without cytopathic changes. The TECs grew well in culture for at least 6 weeks and showed no evidence of infection, cytopathology, or changes in cytokine production with HIV. Only cultures containing macrophages (M phi/TEC or macrophage enriched) showed changes in cytokine production with HIV. Sustained production of IL-1 alpha was seen for up to 20 days, with small or no increases in IL-1 beta. M phi/TEC cultures produced high constitutive levels of IL-6 that were not changed by HIV. Unstimulated macrophage-enriched cultures produced small amounts of IL-6 that were increased by HIV 20-fold. This study suggests that HIV infection in vivo can lead to infection of thymic macrophages resulting in cytokine abnormalities and a constant source for HIV to infect maturing thymocytes. These cytokine effects could lead to abnormal maturation and contribute to the lack of regeneration of the mature CD4+ T cell pool.

HIV-1 infection of macrophages promotes long-term survival and sustained release of interleukins 1 alpha and 6.

HIV infection of macrophages in vivo may result in activation of monokine genes and cause persistent release of immunomodulatory and inflammatory cytokines. Studies that have examined cytokine (IL-1, IL-6, and TNF-alpha) activation by in vitro infection of normal peripheral blood mononuclear cells (PBMCs) with HIV-1 have produced conflicting results. The present study shows that for monokine induction by HIV-1-IIIB preparations derived from the H9 tumor cell line, partial purification of virus particles is essential. Infectious HIV-1 induces the release of high levels of IL-1 alpha, IL-1 beta, and IL-6 bioactivity by adherent PBMCs in the first 3 days following in vitro infection, but only IL-1 alpha and IL-6 continue to be released over several weeks of culture. High levels of bioactive IL-1 beta were released only up to 72 hr following infection, although intracellular IL-1 beta was detectable for at least 3 weeks. No TNF-alpha bioactivity or immunoreactive protein was detectable at > 48 hr in HIV-infected cultures. This time course of monokine release was dependent on the number of infectious particles added to PBMC cultures. In long-term cultures (> 1 month) HIV infection was found to promote the viability of macrophages. The finding of sustained release of IL-1 alpha and IL-6 by infected macrophages, without additional stimulation, suggests that these mediators are released by HIV-1-infected macrophages in AIDS patients, where they may interfere with proper immune regulation.

Interleukin-1 and a 7-kDa T-cell inhibitory monokine reflect disease activity in infection with HIV-1.

Previous studies demonstrated that cultured peripheral blood mononuclear cells (PBMC) from patients with AIDS produce high levels of interleukin 1 (IL-1) and a 7-kDa T-cell inhibitory monokine (TCIM). To determine if the increase in the production of these cytokines corresponded with disease activity, we studied the production of IL-1 and TCIM by PBMC from patients with different stages of human immunodeficiency virus (HIV) infection. Eight patients with asymptomatic seropositive infection, three patients with AIDS-related complex (ARC), three patients with persistent generalized lymphadenopathy (PGL), and six patients meeting the full criteria for diagnosis of AIDS were studied. Patients with AIDS produced increased amounts of TCIM (4.1 times control values, p less than 0.003) and IL-1 (2.0 times control values, p less than 0.05). In contrast, asymptomatic seropositive patients produced less TCIM (0.36 times control values, p less than 0.004) and IL-1 (0.61 times control values, p less than 0.05). Different trends in the levels of these factors produced by patients with ARC and PGL were noted, although results were not statistically significant in general. Patients with ARC tended to produce less IL-1 (0.42 times control values, p less than 0.05), whereas patients with PGL tended to produce increased amounts of IL-1 (1.7 times control values, NS). ARC patients produced a wide range of TCIM values (0.05-2.8 times control values, NS), and patients with PGL tended to produce increased TCIM values, (4.0 times control values, p less than 0.02). No correlations between the levels of IL-1 or TCIM and T-cell subpopulation numbers (CD4 or CD8) or CD4/CD8 ratios were found.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of IL-1 alpha, IL-1 beta, and 25K Mr non-IL-1 activity released by human mononuclear cells.

To determine if the release of IL-1 alpha and IL-1 beta by cultured PBMC could be independently modulated by different exogenous stimuli, we examined the effect of LPS, IFN gamma, latex beads, and indomethacin on the release of IL-1 alpha and IL-1 beta. PBMC culture supernatants were fractionated by Sephacryl-S-200 column chromatography or HPLC (TSK G3000SW), and each fraction was tested for thymocyte mitogenic activity in the presence or absence of preincubation with anti-IL-1 alpha or anti IL-1 beta monoclonal antibody (mAb) and for the presence of IL-1 alpha or IL-1 beta protein by ELISA. In all experiments, thymocyte mitogenic activity not neutralizable by anti-IL-1 alpha or anti-IL-1 beta mAb was detected in the 25K Mr range, which ranged from 12 to 50% of the total thymocyte mitogenic activity released, depending on the stimuli. Cultured PBMC from 95% of individuals release thymocyte mitogenic activity in the absence of exogenous stimuli, which was increased 1.3-to 7-fold by lopopolysaccharide (LPS) (25-50 micrograms/ml). All of this increased activity was due to increased release of IL-1 beta and non-IL-1 thymocyte mitogenic activity, with no change in the total amount of IL-1 alpha released. Indomethacin (0.1 microgram/ml) induced release of increased thymocyte mitogenic activity of 1.3- to 1.4-fold over unstimulated cultures. All of this increased activity was due to increased release of IL-1 alpha and non-IL-1 activity with a concomitant decrease in IL-1 beta release. Interferon gamma (40-100 U/ml) increased the amount of IL-1 alpha and decreased IL-1 beta and non-IL-1 activity released, resulting in no overall change in the total amount of thymocyte mitogenic activity. Molecular weight fractionation of the PBMC culture supernatants revealed that thymocyte mitogenic activity eluting in the 25K Mr range was not due to IL-1 alpha or IL-1 beta. With certain culture conditions, thymocyte mitogenic activity was detected in the 30-40K Mr range. PBMC cultured with LPS and latex beads in the absence of serum released 30-40K Mr IL-1 alpha, as well as 17K Mr IL-1 alpha and 17K Mr IL-1 beta. PBMC cultured in 2% fetal calf serum (FCS) alone from some donors released only 30-40K Mr thymocyte mitogenic activity. Both IL-1 alpha and IL-1 beta protein was detected by ELISA in this Mr range but only the IL-1 alpha was bioactive.(ABSTRACT TRUNCATED AT 250 WORDS)