[Magnetic resonance imaging manifestation of foot injury in amateur half marathon athletes].

Eighty-four amateur half marathon athletes (168 side feet) in Beijing from October 2018 to May 2021 were recruited, and their age, gender and whether they have foot pain were collected, including 44 males and 40 females, aged from 21 to 60 (40.7±9.3) years. All participants underwent bipedal magnetic resonance imaging (MRI) examinations, and the degree of foot pain was graded by foot ankle injury scale (FASS scale). The relationship between MRI features and the foot pain of amateur half marathon athletes were analyzed. The study found that the proportion of foot pain symptoms among amateur half marathon athletes in Beijing was high(122/168), and the MRI manifestations were mainly heel tendinitis and plantar fasciitis, which accounted for about 59.5% of all cases.

Optimization of virus-induced gene silencing in pepper (Capsicum annuum L.).

Virus-induced gene silencing is currently a powerful tool for the study of gene function in plants. Here, we optimized the protocol for virus-induced gene silencing, and investigated factors that affect the efficiency of tobacco rattle virus-induced gene silencing in pepper plants. Consequently, an optimal protocol was obtained by the syringe-infiltration method in the leaves of pepper plants. The protocol involves 2-leaf stage plants, preparing the Agrobacterium inoculum at a final OD600 of 1.0 and then growing the inoculated plants at 22°C. Using this protocol, we achieved high efficiency in silencing CaPDS in different cultivars of pepper plants. We further used the CaPOD gene to illustrate the general reliability of this optimized protocol. Viral symptoms were observed on the leaves of inoculated plants of the Early Calwonder cultivar 25 days post-inoculation, indicating that this protocol can also be used to silence other genes in pepper plants. Real-time polymerase chain reaction analyses revealed that the expression levels of CaPDS and CaPOD were dramatically reduced in inoculated leaves compared to control plants. These results demonstrate that the optimized protocol can be applied to functional genomic studies in pepper to investigate genes involved in a wide range of biological processes.

Genetic determinants of the defense response of resistant and susceptible pepper (Capsicum annuum) cultivars infected with Phytophthora capsici (Oomycetes; Pythiaceae).

Based on culture isolation and morphological observation blight-infected pepper plants in Shaanxi Province, China, we identified the pathogen causing pepper phytophthora blight as Phytophthora capsici. Varieties that differed in resistance (CM334, PBC602, and B27) were inoculated with this pathogen. The root activity of resistant CM334 variety was the highest while that of susceptible B27 variety was the lowest. Also, significant differences in the activity of POD, PAL, and ß-1,3-glucanase were found; there was a positive correlation between disease resistance and activity of these three enzymes. We inhibited mycelial growth and sporangia formation of P. capsici using crude ß-1,3-glucanase and PAL enzymes isolated from the resistant variety CM334 after it had been inoculated with P. capsici. These two enzymes had a synergistic effect on inhibition of P. capsici mycelial growth and sporangia formation. Expression of the defensive genes CaPO1, CaBGLU, CaBPR1, and CaRGA in the three varieties was higher in the leaves than in the roots. All three genes were upregulated in infected leaves and roots of the pepper plants, always expressing at higher levels in the resistant cultivar than in the susceptible cultivar, suggesting that the differences in resistance among the pepper genotypes involve differences in the timing and magnitude of the defense response.

Experimental study on angiogenesis in a rabbit VX2 early liver tumour by perfusion computed tomography.

Ten rabbits implanted with VX2 liver tumours were investigated by perfusion computed tomography (PCT) imaging 1 week (early) and 2 weeks (late) after tumour induction; 10 other rabbits were non-implanted controls. Time-density curves, perfusion parametric maps and perfusion parameters were obtained for tumour rim and normal tissue surrounding the tumour, and for liver tissue from the controls. In addition, microvessel density (MVD) and vascular endothelial growth factor (VEGF) were studied by immunohistochemistry 2 weeks after tumour implantation. A deconvolution mathematical model was used to calculate hepatic blood flow (HBF), hepatic blood volume (HBV), mean transit time (MTT), capillary vessel surface permeability (PS) and hepatic arterial index (HAI). At the tumour rim on the early PCT scan, MTT was significantly lower whereas HBF, HBV, HAI and PS were significantly higher than in surrounding normal tissue. There were no significant changes in perfusion parameters on the late PCT scan compared with the early scan. Significant linear correlations of MVD and VEGF were found with HBF, PS and HAI, but not with HBV or MTT. It is concluded that PCT imaging is useful for the evaluation of tumour angiogenesis and for the early detection of liver tumours.

The synthetic liver X receptor agonist GW3965 reduces tissue factor production and inflammatory responses in human islets in vitro.

AIMS/HYPOTHESIS: Optimising islet culture conditions may be one strategy for reducing islet loss prior to, and immediately after, islet transplantation. Liver X receptor (LXR) agonism has previously been shown to increase insulin release from pancreatic islets and reduce inflammation in leucocytes. Our aim was to investigate whether the synthetic LXR agonist GW3965 could modulate the inflammatory status of human pancreatic islets. METHODS: Levels of pro-inflammatory cytokines and tissue factor in isolated human islets were determined by TaqMan low density array and/or real-time quantitative RT-PCR (mRNA levels) and enzyme immunoassay (EIA) (protein levels). Islet viability was measured using intracellular ATP content, ADP/ATP ratio, mitochondrial dehydrogenase activity (XTT assay) and insulin secretion in a dynamic glucose-challenge model. Apoptosis was determined by EIA measurement of histone-DNA complexes present in cytoplasm and by assaying caspase-3/-7 activity. RESULTS: Treatment of LPS-stimulated human islets with the synthetic LXR agonist GW3965 (1 micromol/l) for 24 h reduced mRNA and protein levels of selected pro-inflammatory cytokines (IL-8, monocyte chemotactic protein-1 and tissue factor). Moreover, GW3965 had no adverse effect on insulin secretion, islet viability or apoptosis. No excess of lipid accumulation could be detected with the dosage and exposure time used. CONCLUSIONS/INTERPRETATION: LXR activation suppresses inflammation in human islets in vitro without adverse effects on islet viability. Short-term moderate activation of LXR prior to islet transplantation may represent a possible strategy for improving post-transplant islet survival.

Residual bodies and IL-1alpha stimulate expression of mRNA for IL-1alpha and IL-1 receptor type I in cultured rat Sertoli cells.

The cytokine interleukin (IL)-1alpha may be produced both by Sertoli cells and immature male germ cells from rat and is thought to play a role in autocrine and/or paracrine regulation of the spermatogenesis. The localization of IL-1 receptors in seminiferous tubules is unknown. In this study we found a constitutive expression of IL-1 receptor type I (IL-I RI) mRNA in cultured Sertoli cells and peritubular cells from rat, whereas no such transcripts were observed in immature germ cells (pachytene spermatocytes and round spermatids). An autostimulation of IL-1alpha mRNA synthesis has previously been described in other cell types. Stimulation of Sertoli cells with recombinant IL-1alpha for 0-7 h resulted in a rapid increase in both IL-1alpha and IL-1 RI mRNA. When Sertoli cells were cultured with residual bodies for 0-48 h, mRNA levels for both IL-1alpha and IL-1 RI were increased in a biphasic manner. We suggest that phagocytosis of residual bodies triggers an autocrine IL-1alpha loop in Sertoli cells where both IL-1alpha and one of its receptors are stimulated.

Peptidoglycan primes for LPS-induced release of proinflammatory cytokines in whole human blood.

The pathophysiological mechanisms involved in mixed bacterial infections caused by gram-positive and gram-negative bacteria are largely unknown. The present study examines the potential interaction between lipopolysaccharide (LPS) and peptidoglycan (PepG) in the induction of the sepsis-associated cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-10 in whole human blood. Plasma values of these cytokines were measured by enzyme immunoassays and a TNF bioassay. Co-administration of PepG (10 microg/mL) or muramyl dipeptide (MDP, 1 microg/mL) with LPS (10 ng/mL) caused significantly elevated values of TNF-alpha and IL-6 in the blood that could not be obtained by the sum of the values obtained by each stimulant alone, or by 3-fold higher doses of either bacterial component alone. This phenomenon was observed 1 h after stimulation, throughout the experimental period (24 h), and with different doses of LPS and PepG. In contrast, the release of IL-10 was not influenced by the co-administration of PepG or MDP with LPS. The TNF-alpha release induced by co-administration of LPS and PepG was abrogated after pretreatment with a monoclonal antibody against CD14 (18D11). Addition of PepG or MDP to whole blood caused a 2-fold increase in the surface expression of CD14 on monocytes, as measured by flow cytometry. In contrast, LPS caused decreased expression of this receptor. Our data suggest that PepG and MDP primes human whole blood leukocytes for LPS-induced release of proinflammatory cytokines. We speculate that synergy between PepG and LPS may contribute to the pathogenesis in sepsis caused by mixed bacterial infections.

Procalcitonin does not influence the surface expression of inflammatory receptors on whole blood leukocytes.

The plasma levels of procalcitonin (PCT) are increased in patients with severe bacterial infections. Its cellular origin and potential pathophysiological function in sepsis is, however, unclear. White blood cells have recently been described to express both PCT mRNA and protein. The aim of this study was to determine whether PCT has any influence on the surface expression of receptors, relevant in inflammation, on human whole blood leukocytes under normal and septic conditions. Venous blood from healthy donors was incubated with PCT (40 ng/ml or 1200 ng/ml) alone or in combination with lipopolysaccharide (LPS, 10 ng/ml) or peptidoglycan (PepG, 10 micrograms/ml) for 6 h. The surface expression of CD14, CD54, CD64, CD80, CD86 and HLA-DR was determined by flow cytometry. We could not detect any influence of PCT on the expression of these receptors. Further studies on potential effects on other cell types during infection seem warranted.

Hydrogen peroxide contrast hepatosonography.

Hydrogen peroxide contrast hepatosonography (HPCH), a new technique, was developed for liver examination. On examination, 5-10 ml of 2-3% hydrogen peroxide was injected into the rectum through a dual-channel rubber tube. When hydrogen peroxide passed through the mucosa into the portal vein, the fast flowing contrast echoes were seen, and the liver parenchyma was covered by enhanced echoes. A total of 297 subjects were examined by HPCH with a successful contrast rate of 95.6%. In normal subjects, dense contrast echoes were visible throughout the liver. But in patients with hepatoma, hemangioma, cyst and abscess, contrast echo defects were noted. We conclude that HPCE is of great value in the differentiation of space-occupying lesions and measurement of portal circulation time and blood flow velocity in spite of its untoward reactions.

Application of two-dimensional echocardiography in location of balloon of the Swan-Ganz catheter.

Two-dimensional echocardiography was used to guide and locate the balloon of Swan-Ganz catheter during catheterization in 43 patients. The echoes of balloon appeared as a series of strong light spots, with a rate of detection of 100%. Under monitoring of two-dimensional echocardiography, the site of balloon and its moving route could be determined. The coincidence of echographic findings and X-ray findings showed that it can be used instead of X-ray monitoring to lessen irradiation during catheterization. The depth of catheter inserted was measured. The differential points between the echoes of balloon and tubing, the terminology of the balloon site in echocardiography and fluoroscopy, its clinical value and limitation are discussed.

Oral and systemic administration of beta-glucan protects against lipopolysaccharide-induced shock and organ injury in rats.

beta-Glucans are glucose polymers with a variety of stimulatory effects on the immune system. The objective of this study was to determine the effect of prophylactic oral administration of soluble Saccharomyces cerevisiae-derived beta-1,3/1,6-glucan (SBG) on the outcome of experimental endotoxaemia and shock-associated organ injury. Male Wistar rats were pretreated with SBG orally (SBGpo, 20 mg/kg/day) for 14 days, subcutaneously (SBGsc, 2 mg/kg/day) for 3 days, or vehicle (placebo). Rats were anaesthetized and subjected to endotoxaemia by intravenous infusion of Escherichia coli lipopolysaccharide (LPS) (6 mg/kg) or saline infusion (sham). We observed significant levels of plasma beta-glucan in the SBGpo group (P<0 x 5), although the SBGsc group had levels approximately 40-fold higher despite a 10-fold lower dose. SBG prophylaxis caused enhanced blood pressure recovery following LPS-induced blood pressure collapse. Oral treatment with SBG attenuated the LPS-induced rise in plasma creatinine levels (P<0 x 05), indicating protection against renal injury. SBG also attenuated the plasma levels of aspartate aminotransferase and alanine aminotransferase (SBGpo, P<0 x 01; SBGsc, P<0 x 01), indicating protection against LPS-induced hepatic injury. A moderate increase in baseline interleukin (IL)-1beta levels was observed in the SBGsc group (P< 0 x 05). In the LPS-challenged rats, plasma levels of proinflammatory cytokines was moderately reduced in both SBG-treated groups compared to placebo. SBG treatment, particularly oral administration, had a striking effect on the haemodynamics of LPS-treated rats, although only a minute fraction of the orally administered beta-glucan translocated to the circulation. Enhanced organ perfusion may thus be responsible for the attenuated levels of indicators of kidney and liver injury seen in SBG-treated rats.

Cytokine responses to CpG DNA in human leukocytes.

Previous studies have implicated a role of bacterial DNA, containing unmethylated cytosine-phosphate-guanosine (CpG) motifs, in the initiation of systemic inflammation. This is based on the ability of CpG-DNA to act in synergy with lipopolysaccharide (LPS) to trigger tumor necrosis factor alpha (TNFalpha) production in murine monocytes and to enhance LPS toxicity in rodents. In this study we investigated the capacity of CpG-DNA to trigger and modulate cytokine responses in human leukocytes. A human blood assay, as well as isolated cultures of monocytes and neutrophils, was exposed to the synthetic oligodeoxynucleotides (ODNs) CpG ODN (2006) and GpC ODN (2006-GC), alone or in combination with peptidoglycan or LPS. Plasma or supernatants were isolated and analyzed for TNFalpha, interleukin-1 beta (IL-1beta), IL-6 and IL-8 by ELISA. In the blood, 2006 (but not 2006-GC) induced the release of TNFalpha (P < 0.05) and possibly IL-1beta and IL-6. IL-8 was induced in a CpG-independent manner. When co-administered with peptidoglycan, both ODNs enhanced the release of cytokines, but not consistently CpG dependent. When co-administered with LPS, only IL-8 values were enhanced, whereas IL-6 was suppressed at early time points. In monocyte and neutrophil cultures, CpG dependent induction of cytokine release was not observed. However, both ODNs inhibited LPS-induced IL-6. In conclusion, the capacity of CpG DNA to trigger the release of TNFalpha and to enhance LPS-induced release of this cytokine is confirmed in human whole blood, but not in adherent human monocytes. Most effects of the ODNs on cytokine release in human leukocytes were CpG independent.

Anti-inflammatory properties of enamel matrix derivative in human blood.

BACKGROUND AND OBJECTIVE: Enamel matrix derivative (EMD), extracted from porcine tooth buds, has been shown to promote periodontal healing in patients with severe periodontitis. This involves modulation of the inflammatory response followed by the onset of periodontal regeneration. Based on these observations, we examined the ability of EMD to modulate the release of a pro-inflammatory cytokine [tumor necrosis factor (TNF)-alpha], an anti-inflammatory cytokine (interleukin-10) and a chemokine (interleukin- 8) in whole human blood challenged by bacterial cell wall components. MATERIAL AND METHODS: Whole blood from healthy donors was challenged by lipopolysaccharide or peptidoglycan and incubated with different concentrations of EMD or a cAMP analogue 8-(4-chlorophenyl)thio-cAMP (8-CPT-cAMP). TNF-alpha, interleukin-8 and interleukin-10 were analysed from plasma by enzyme-linked immunosorbent assay (ELISA) while cAMP levels of peripheral blood mononuclear cell lysates were analysed by enzyme immunoassay (EIA). RESULTS: We found that EMD attenuated the release of TNF-alpha and interleukin-8 in whole blood from healthy donors challenged by lipopolysaccharide or peptidoglycan, while the release of interleukin-10 was unchanged. Enamel matrix derivative also produced a four-fold increase in the cAMP levels of peripheral blood mononuclear cell lysates. Like EMD, 8-CPT-cAMP attenuated the formation of TNF-alpha, but not of interleukin-10, in blood challenged by lipopolysaccharide. CONCLUSION: Enamel matrix derivative limits the release of pro-inflammatory cytokines induced by lipopolysaccharide or peptidoglycan in human blood, suggesting that it has anti-inflammatory properties. We propose that this effect of EMD is, at least partly, secondary to an increase in the intracellular levels of cAMP in peripheral blood mononuclear cells.

Reduced capacity of whole blood leucocytes to express tumour necrosis factor-alpha and interleukin-10 following major orthopaedic surgery.

BACKGROUND: Severe trauma is a challenge to the immune response and may cause reduced immune capacity. As a marker of decreased cellular activity, studies with ex vivo lipopolysaccharide (LPS) stimulation of whole blood or isolated mononuclear cells from injured patients have revealed reduced production of inflammatory cytokines. To gain further insight into immune alterations in orthopaedic surgery, we studied LPS-induced tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 in whole blood of patients during peri- and postoperative phases of total hip replacement. METHODS: Four females and 3 males undergoing elective total hip replacement were included in the study. Ex vivo LPS-induced TNF-alpha and IL-10 were measured in a whole blood assay before, during and at 1 and 6 days after operation. In addition, the counts of white blood cells were determined. RESULTS: During the operation, there were significant reductions in the number of monocytes, but at day 1 and 6 after surgery, there were significant increases as compared to the levels before surgery. The capacity of whole blood to express TNF-alpha and IL-10 did not change significantly during the operation and the following postoperative day. At day 6, however, there were significant reductions in expression of both TNF-alpha and IL-10 as compared to the levels before the operation. In relation to the values of monocytes, there was a significant reduction in the expression of TNF-alpha also at day 1 after operation. CONCLUSION: Our data indicate that in the course of at least 6 days after a major orthopaedic trauma, there is suppression of the whole blood capacity to express the inflammatory cytokine TNF-alpha and the anti-inflammatory cytokine IL-10 when exposed to LPS. During this time, then, the patient is particular susceptible to septic complications.

Peptidoglycan of Staphylococcus aureus upregulates monocyte expression of CD14, Toll-like receptor 2 (TLR2), and TLR4 in human blood: possible implications for priming of lipopolysaccharide signaling.

Previous studies have indicated that peptidoglycan (PepG) from gram-positive bacteria can exert a priming effect on the innate immune response to lipopolysaccharide (LPS) from gram-negative bacteria. Here, we hypothesized that this priming effect may be preceded by enhanced expression of monocyte CD14, Toll-like receptor 2 (TLR2), and TLR4. In an ex vivo whole human blood model, we observed a substantial synergy between LPS and PepG in the release of tumor necrosis factor alpha and interleukin-1beta (IL-1beta) over the 24-h experimental period, whereas the effect on IL-8 and IL-10 release was more time dependent. The priming effect of PepG on cytokine release was preceded by a rapid upregulation of CD14, TLR2, and TLR4 expression on monocytes: at 3 hours there was a twofold increase in CD14 expression (P < 0.03), a fivefold increase in TLR2 expression (P < 0.03), and a twofold increase in TLR4 expression (P < 0.03). CD14 and TLR2 remained upregulated throughout the experimental period following exposure to PepG (P < 0.05). Only a transient upregulation of these monocyte receptors was observed following treatment with LPS or LPS plus PepG. In conclusion, the synergistic effect of LPS and PepG on cytokine release is preceded by a reciprocal upregulation of TLR2 and TLR4 by both bacterial cell wall components.

Cytokine responses to fungal pathogens in Kupffer Cells are Toll-like receptor 4 independent and mediated by tyrosine kinases.

Disseminated fungal infections are increasing. However, the interactions between the body's largest population of tissue macrophages, the Kupffer cells and the fungal pathogens are scarcely understood. The aim of this study was to examine the involvement of Toll-like receptor 4 (TLR4) signalling in cytokine production, using primary cultures of rat and murine Kupffer cells exposed to Aspergillus fumigatus and Candida albicans hyphae and conidia. All fungal components induced the release of tumour necrosis factor-alpha (TNF-alpha), but with delayed kinetics compared with lipopolysaccharide (LPS). Candida albicans was the most potent inducer of TNF-alpha protein and mRNA and the only inducer of interleukin-10 (IL-10) in rat Kupffer cells. All fungal components induced enhanced mRNA levels of macrophage inhibitory protein-2 (MIP-2) in the cells, similar to LPS. Inhibitors of Src tyrosine kinases added to cells prior to stimulation led to attenuation in the release of both TNF-alpha (60%, P < 0.05) and IL-10 (70%, P < 0.05) induced by C. albicans conidia but did not influence the LPS-mediated cytokine release. Murine Kupffer cells (C57BL/10J) also released TNF-alpha as well as the chemokines keratinocyte-derived chemokine (KC) and MIP-2 in response to fungal component. Surprisingly, Kupffer cells from TLR4-deficient C57BL/ScCr mice exhibited significantly enhanced production of KC and MIP-2 upon stimulation by fungal components compared with control littermates (P < 0.05). Our study demonstrates that Aspergillus and Candida components induce cytokine production in rat Kupffer cells and that the response to C. albicans conidia involves Src tyrosine kinases. The experiments with TLR4-deficient Kupffer cells suggest that the cytokine response in these cells to fungal component is not mediated by TLR4.

A study on the epidemiology of hepatitis C infection among blood donors in Singapore.

BACKGROUND: Hepatitis C viral (HCV) infection has been identified as the main cause of post-transfusion hepatitis (PTH) since 1989. Despite this, little is known regarding the prevalence and mode of transmission of the disease. The purpose of this study was therefore to study the demographic factors associated with HCV infection among Singaporean blood donors. METHODS: In this study, the screening questionnaire records of HCV-positive donors were analysed. A total of 241 donors, tested positive for HCV between 7 December 1992 and 31 August 1994, were included. Demographic details studied included the age, sex, race, citizenship, occupation and number of previous donations. In additions, the associations of HCV infection with other screened diseases were analysed. RESULTS: The prevalence of HCV infection was found to be 0.370 per cent (241/65208) among the donors. Of these, 200 (0.389 per cent prevalence) were male and 41 (0.298 per cent prevalence) were female. The mean age was 34.2, SD = 9.4. The prevalence of the disease was found to increase with age. Significant differences were seen among the races (chinese versus Malay, 0.329 percent versus 0.513 percent, p < 0.05). There was also a significant association of HCV with Human Immunodeficiency Virus (HIV) infection among the donors (0.4 percent co-infection versus 0.004 percent in the general donor population, p < 0.01). CONCLUSION: The incidence of HCV infections is relatively low among blood donors in Singapore. The differences in prevalence seen among the different groups studied suggest that the disease is community acquired and may be due to the lifestyle of the donors.

PIP: A retrospective review of the screening records of the 65,208 blood donors in Singapore from December 7, 1992, to August 31, 1994, revealed a relatively low prevalence of hepatitis C infection. Hepatitis C is the main cause of post-transfusion hepatitis. A total of 241 donors were positive for this strain, for a period prevalence of 0.37%. 58% of the hepatitis C-positive donors were repeat donors. Despite the low overall prevalence of hepatitis C infection, certain demographic patterns emerged among seropositive donors. Most cases involved men in the 30-39 year age group. In terms of occupations, seropositive donors were fairly equally distributed among the production and labor workforce, sales and services, managerial and professional posts, and the police and armed forces. Although there was no significant difference between the general donor population and hepatitis C-infected donors in terms of hepatitis B or syphilis infection, the latter were significantly (p 0.01) more likely to be infected with human immunodeficiency virus (HIV). Finally, hepatitis C infection was significantly (p 0.05) more prevalent in Malay donors than in Chinese. The findings suggest that hepatitis C is community acquired. Although Singapore as a whole is assumed to have a low incidence of hepatitis C, generalizations cannot be made from this study given the stringent criteria for selection as a blood donor.

Response of alkalinization or acidification by phytohemagglutinin is dependent on the activity of protein kinase C in human peripheral T Cells.

The increase of intracellular free calcium concentration ([Ca(2+)](i)) and protein kinase C (PKC) activity are two major early mitogenic signals to initiate proliferation of human T cells. However, a rapid change in intracellular pH (pH(i)), acidification or alkalinization during the activation, is also associated after these two signals. The aim of this study was to define whether the change in pH(i) is affected by calcium and protein kinase C (PKC), in phytohemagglutinin (PHA)-stimulated T cells. T cells were isolated from human peripheral blood. The [Ca(2+)](i) and the pH(i) were measured using, respectively, the fluorescent dyes, Fura-2, and BCECF. In addition, down-regulation of PKC activity by PMA (1 microM, 18 h) was confirmed in these cells using a protein kinase assay. The results indicated that, (1) alkalinization was induced by PHA or PMA in T cells; the results of alkalinization was PKC-dependent and Ca(2+)-independent, (2) in PKC down-regulated T cells, PHA induced acidification; this effect was enhanced by pre-treating the cells with the Na(+)/H(+) exchange inhibitor, 5-(N,N-dimethyl)-amiloride, (DMA, 10 microM, 20 min), (3) the acidification was dependent on the Ca(2+) influx and blocked by removal of extracellular calcium or the addition of the inorganic channel blocker, Ni(2+), and (4) Thapsigargin (TG), a Ca(2+)-ATPase inhibitor, confirmed that acidification by the Ca(2+) influx occurred in T cells in which PKC was not down-regulated. These findings indicate two mechanisms, alkalinization by PKC and acidification by Ca(2+) influx, exist in regulating pH(i) in T cells. This is the first report that PHA stimulates the acidification by Ca(2+) influx but not alkalinization in T cells after down-regulation of PKC. In conclusion, the activity of PKC in T cells determines the response in alkalinization or acidification by PHA.