Peripheral Mononuclear Cell Cultures Research Articles

The impact of SARS-CoV-2 peptides on activation of NK cells

BACKGROUND: NK cells, alone with T lymphocytes, have a high antiviral activity. Exploring the contribution of NK cells in fighting SARS-CoV-2 infection may promote the development of appropriate treatments for COVID-19. Previously, NK cell response was considered nonspecific, provided by a combination of signals from activating and inhibitory receptors. Currently, the existence of certain subpopulations of antigen-specific, or adaptive, NK cells has been shown.
 AIM: To evaluate the functional response of NK cells induced by SARS-CoV-2 peptides.
 MATERIALS AND METHODS: The functional response of NK cells to SARS-CoV-2 peptides was determined by their degranulation (surface CD107a expression) and IFN production levels, and by the activation degree (HLA-DR expression level). Volunteers who recovered from COVID-19 participated in the study, and immune cells from a healthy volunteer without SARS-CoV-2-specific antibodies were used as controls.
 RESULTS: NK cells from individuals who had recovered from COVID-19, in contrast to a donor who had not been infected, showed a higher level of IFN production in response to SARS-CoV-2 peptides, compared with control samples. The level of degranulation of NK cells from donors previously infected with SARS-CoV-2 was higher than in the corresponding control. The proportion of activated NK cells obtained from recovered donors was also higher in samples stimulated with SARS-CoV-2 peptides.
 CONCLUSIONS: We have demonstrated the activation of NK cells obtained from people who had previously recovered from COVID-19 in response to SARS-CoV-2 peptide antigens in cultures of peripheral mononuclear cells in vitro. This study reveals the possibility for further investigation of antigen-specific NK cells in COVID-19 disease. The use of such cells could help develop treatments for SARS-CoV-2 infection.

PAR1‐stimulated platelet releasate promotes angiogenic activities of endothelial progenitor cells more potently than PAR4‐stimulated platelet releasate

Endothelial progenitor cells (EPCs) are important for endothelial regeneration and angiogenesis. Thrombin protease-activated receptor 1 (PAR1) PAR1 and PAR4 stimulation induces selective release of platelet proangiogenic and antiangiogenic regulators. To investigate if PAR1-stimulated platelet releasate (PAR1-PR) and PAR4-PR regulate angiogenic properties of EPCs in different manners. EPCs were generated from peripheral mononuclear cell culture. Washed platelets (2 × 10(9) mL(-1)) were stimulated by PAR1-activating peptide (PAR1-AP; 10 μmol L(-1)) or PAR4-AP (100 μmol L(-1)) to prepare PAR1-PR and PAR4-PR, respectively. PAR1-PR or PAR4-PR had little influence on EPC proliferation. EPC migration experiments using a modified Boyden chamber showed that both platelet releasates facilitated EPC migration. As for in vitro tube formation on Matrigel, PAR1-PR and PAR4-PR similarly enhanced capillary-like network formation of EPCs in the complete EPC medium containing 10% FBS and a cocktail of growth factors, while PAR1-PR more profoundly increased EPC tube formation in basal culture medium supplemented with only 0.5% FBS than did PAR4-PR. The latter was confirmed in the murine angiogenesis model of subcutaneous Matrigel implantation. Moreover, blockade of vascular endothelial growth factor, stromal cell-derived factor 1α, or matrix metalloproteinases attenuated EPC migration and tube formation, suggesting a cooperation of these factors in the enhancements. PAR1-PR enhances vasculogenesis more potently than PAR4-PR, and the enhancements require a cooperation of multiple platelet-derived angiogenic regulators.

Evaluation of Th17 related cytokines associated with clinical and laboratorial parameters in sickle cell anemia patients with leg ulcers

Leg ulcers (LUs) represent one of the main causes of morbidity in sickle cell anemia (SCA). This manifestation has been related to hemolysis, infections predisposition and inflammation that leads cytokines secretion. In this context, our study aimed to evaluate Th17 related cytokines (IL-6, IL-17A, IL-22 and IL-23) in serum and peripheral mononuclear cells culture supernatants with and without lymphoproliferative stimulation (anti-human CD3 and anti-human CD28). The cytokines levels were also correlated to clinical, hematological and biochemical parameters in SCA patients with and without LUs history (SCALU and SCAWH) as well as in healthy controls. In SCALU patients, high levels of IL-17A were associated with absence of acute chest syndrome (ACS, p=0.0328). The other clinical parameters analyzed (osteonecrosis, stroke, priapism, splenectomy and blood transfusions history) were not significantly related with other cytokine levels. In SCALU patients was also observed that IL-17A increased levels were associated with high levels of LDH (p=0.0130), the same association pattern was found for IL-6 (0.0160) and IL-22 (p=0.0165) in the SCALU group. Interestingly, we did not find statistical correlations with these parameters in SCAWH group. The other hematological parameters (hemoglobin, leucocyte and reticulocyte count) and indirect bilirrubin did not show any correlation with analyzed cytokines in both groups. So, for the first time, we show that IL-17A present in SCALU patients may exert a preventive role in the ACS development. Furthermore, IL-6, IL-17A and IL-22 accompanied the LDH levels only in SCALU patients suggesting to serve as additional markers of hemolysis or to be related with immunity response against extracellular pathogens.

Heat shock protein 60 from Klebsiella pneumoniae protects mononuclear cells from apoptotic cell death induced by dexamethasone

AimsBacterial heat shock proteins can have anti-apoptotic effects on human cells. We investigated whether enterobacterial HSP60 can protect peripheral blood mononuclear cells (PBMC) from DXM-induced apoptosis and if this effect requires cytoskeleton participation. Main methodsAnti-apoptotic effect from enterobacterial HSP60 was analyzed by adding these proteins to peripheral mononuclear cells cultures before DXM induction. Percentage of apoptotic cells was determined by SubG0 peak and TUNEL techniques in a flow cytometer. Key findingsOur results showed significant protective effect of HSP60 Klebsiella pneumoniae and E. coli, in the DXM-induced apoptosis in PBMC. Similar results were obtained with recombinant human HSP60. The same protective effect of proteins was observed in CD4+ and CD8 + T cell subpopulations. To analyze if enterobacterial HSP60 need internalization to have the anti-apoptotic effect, we used cytoskeleton inhibitors such as: nocodazole, cytochalasin D and amiloride, the three inhibitors significantly affected the protective role of HSP60 in apoptosis induced with DXM. Results suggest that the protective effect of HSP60 K. pneumoniae and E. coli requires the participation of contractile structures for the internalization of this protein by the cells, we suggest that the internalization of enterobacterial HSP60 could be carry out by macropinocytosis. SignificanceWe report for the first time that K. pneumoniae and E. coli HSP60 have protective effect in the apoptosis induced with DXM in PBMC from healthy subjects and that this effect requires the internalization of the protein with active participation of the cytoskeleton.

In Vitro Cytokine Responses to Periodontal Pathogens: Generalized Aggressive Periodontitis is Associated with Increased IL-6 Response to Porphyromonas gingivalis

Generalized aggressive periodontitis (GAgP) is an inflammatory condition resulting in destruction of tooth-supporting tissues. We examined the production of IL-1beta, IL-6, tumour necrosis factor (TNF)-alpha, IL-12 and IL-10 in cultures of peripheral mononuclear cells (MNC) from 10 patients with GAgP and 10 controls stimulated with periodontal pathogens or a control antigen, tetanus toxoid (TT) in the presence of autologous serum. The pathogens used were Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum, either as type strains or bacteria isolated from the participants' inherent oral flora. The P. gingivalis -induced production of IL-6 was approximately 2.5-fold higher in patients with GAgP than in healthy controls (P < 0.05), while the corresponding TNF-alpha production was non-significantly elevated. IL-1beta production induced by P. gingivalis, as all cytokine responses induced by Pr. intermedia, F. nucleatum and TT was similar in the two groups. A reduced IL-12p70 response to Pr. intermedia and F. nucleatum was observed in smokers compared to non-smoking patients (P < 0.02). To assess the role of serum factors in the elevated IL-6 response to P. gingivalis, MNC from two donors free of disease were stimulated with this bacterium in the presence of the various patient and control sera. An elevated IL-6 and TNF-alpha response was observed in the presence of patient sera (P < 0.01 and P < 0.04, respectively). The data suggest that an exaggerated production of IL-6 occurs in GAgP, and that pro-inflammatory serum factors play an essential role in the response.

Specific antibody-forming cells in bronchoalveolar lavage fluid of patients with summer-type hypersensitivity pneumonitis--detection by enzyme-linked immunospot (ELISPOT)

Antigen-specific antibody-forming cells (AFCs) were assessed in bronchoalveolar lavage (BAL) fluid of patients with summer-type hypersensitivity pneumonitis by the method of enzyme-linked immunospot (ELISPOT). Both Cryptococcus- and Trichosporon-specific AFCs of isotypes of IgG, IgA and IgM were detected in BAL fluid cells. The frequency of isotypes of AFCs was in the order of IgA > IgM > IgG. Antibody activities against both Cryptococcus and Trichosporon antigens were detected in the culture supernatant of BAL cells. A good correlation was observed between the number of AFCs and the degree of antibody activity of culture supernatant of BAL cells, particularly of IgA isotype. For the IgM and IgA isotypes, anti-Cryptococcus antibody activity correlated well with anti-Trichosporon antibody activity (r = 0.98, p < 0.0001). In several cases examined, antigen-specific AFCs were detected in in vitro culture of peripheral blood mononuclear cells of patients after stimulation with PWM. In the present study, we demonstrated that Cryptococcus neoformans and Trichosporon cutaneum antigen-specific AFCs were present in the lungs of patients with summer-type hypersensitivity pneumonitis, and the antibodies secreted therefrom reacted with both these antigens. The presence of memory B cells was also suggested in the peripheral blood of patients.

Buprenorphine and methadone maintenance treatment of heroin addicts preserves immune function

Opiate addiction influences many physiological functions including immune responses. The objective of this study was to investigate the immune system function in heroin addicted patients submitted to methadone or buprenorphine maintenance treatment compared to untreated heroin addicts and healthy controls.Four groups were studied: group A included nine heroin addicted subjects, who were still injecting heroin; groups B and C were composed of 12 patients previously addicted to heroin, being treated with methadone (mean dosage 58±12.7mg/day) or buprenorphine (mean dose 9.3±2.3mg/day) since at least 6 months; group D was composed of 15 sex and age matched healthy controls. Lymphoproliferation and peripheral mononuclear cell cultures production of the Th1 cytokines IL-2 and IFN-γ, the Th2 cytokine IL-4, and of the pro-inflammatory cytokine TNF-α were evaluated in all the patients and controls.PHA-lymphoproliferation was lower in untreated heroin addicts than in controls, while it was normal in methadone and buprenorphine treated patients. An altered Th1/Th2 balance, characterized by reduced IL-4, IFN-γ and TNF-α but normal IL-2 levels, was present in untreated heroin addicted subjects, while the Th1/Th2 balance was well conserved in the methadone and buprenorphine groups.These findings suggest that the immune system abnormalities in heroin addicted patients can be restored to almost normal values by controlled treatment with methadone and buprenorphine.

Serial analyses of viral levels and immunocytes during in vitro incubation of peripheral blood mononuclear cells from HIV-infected individuals

To investigate the dynamic changes of viral loads and immunocytes during the in vitro culture of peripheral blood mononuclear cells (PBMC) from HIV carriers. The PBMCs from 14 HIV-infected individuals and 6 healthy persons were incubated in serum-free AIM-V medium containing cocktail cytokines. The phenotype of CD3, CD4, CD8, CD3CD56 and CD25 was identified by flow cytometric analysis every two days. The production of cytokines in the supernatants, including IL-1alpha, IL-12, TNF-alpha and IL-10 was measured by ELISA. The supernatant HIV-1 RNA load was also determined by Real-time fluorescent PCR. During a 21-day incubation period, The PBMCs multiplied approximately 60.7-fold and 16.8-fold respectively in the healthy controls and 7 out of the 14 HIV-infected subjects, however failed to multiply in the remaining 7 HIV-infected subjects. The expanded cells were phenotypically shown a heterogeneous cellular population with 23.3%-35% for CD3(+)CD4(+) T cells and 58.7%-72% for CD3(+)CD8(+) T cells, and approximate 17% CD3(+)CD56(+) cells at 16-day incubation for HIV-infected cases. HIV-1-positive PBMCs were found to produce an elevated ratios (value range 6.01 - 48.04) of IL-12:IL-10 compared to healthy individuals (6.65 - 10.2) at 16-day incubation. Furthermore, serial analyses of HIV-1 RNA levels showed an inverted V type dynamic change during 16 day in vitro incubation period. In vitro expansion of functional immunocytes of HIV-1 carrier origin is feasible and may facilitate the autologous antiviral immune therapy for HIV-infected patients.

Th-1 and Th-2 cytokine production in infants with virus-associated wheezing

Wheezing associated with respiratory viral infections in infancy is very common and results in high morbidity worldwide. The Th1/Th2 pattern of immune response in these patients remains unclear and previous studies have shown controversial results. The aim of the present study was to compare the type of Th1/Th2 cytokine response between infants with acute bronchiolitis, recurrent wheezing and upper respiratory infections from a developing country. Infants younger than 2 years of age admitted to Hospital São Lucas, Porto Alegre, RS, Brazil, between May and November 2001, with an acute episode of wheezing associated with viral respiratory infection were selected. Subjects with upper respiratory infections from the emergency department were selected for the control group. Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) levels from nasal aspirates were determined by ELISA from peripheral mononuclear cell cultures. Twenty-nine subjects with acute bronchiolitis, 18 with recurrent wheezing and 15 with upper respiratory infections were enrolled. There were no differences in family history of atopy or parental smoking between groups. Oxygen requirement was similar for the acute bronchiolitis and recurrent wheezing groups. The percentage of positive tests for the cytokines studied and the IFN-gamma/IL-4 ratio was similar for all groups. Comparison of the polarized Th1/Th2 cytokine results for the various groups showed no specific pattern of cytokine production. Infants with wheezing from a developing country do not show any specific predominant pattern of Th1/Th2 cytokine production, suggesting that multiple factors may be involved in the pathogenesis of this illness.

Induction of Nitric Oxide Production Mediated by Tumor Necrosis Factor Alpha on Staphylococcal Enterotoxin C-Stimulated Bovine Mammary Gland Cells

Mammary gland (MG) secretions (MGS) derived from secretory cows infected with coagulase-negative staphylococci (CoNS) showed somatic cell counts and lactoferrin similar to levels found in the MGS of secretory cows infected with Staphylococcus aureus. However, nitrite and nitrate (NOx) and staphylococcal enterotoxin C (SEC) were found in MGS infected with S. aureus at much higher levels than in cows infected with CoNS. These results suggested that NOx could be intimately correlated with the production of SEC in secretory cows infected with S. aureus. Therefore, we examined the production of NOx and the expression of proinflammatory cytokines and microsomal cytochrome P450 (CYP450) after injection of SEC into the MGS of secretory cows. We were able to detect NOx and the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) on MG cells of SEC-injected MGS. It was also found that CYP450 in the MG cells from SEC-injected MGS was down-regulated by approximately one-third in comparison with the cells from phosphate-buffered saline-injected MGS. This in vitro system also showed that NOx could be induced in the culture of bovine macrophage-lined cells (FBM-17) with the supernatants of SEC-stimulated bovine peripheral blood lymphocytes (BoPBLs) but not in the culture of peripheral mononuclear cells with SEC-stimulated BoPBLs. The expression of the mRNA for both inducible nitric oxide synthase and TNF-alpha in FBM-17 was enhanced by culturing with the supernatant of SEC-stimulated BoPBLs, although CYP450 was down-regulated. These results indicate that the down-regulation of CYP450 was caused by the production of TNF-alpha in SEC-stimulating MG cells containing macrophages and via NOx production. Therefore, we suggest that NOx released from activated MG cells via the superantigenic activity of SEC caused oxidative damage to the MG in S. aureus-induced mastitis.

Effect of lymphokines on 35sulfate uptake by the glomerular basement membrane.

We have previously shown that lymphocytes from idiopathic minimal-lesion nephrotic patients produce a lymphokine (supernatant factor) that increases the 35sulfate uptake in the glomerular basement membrane (GBM). The purpose of this report was to further characterize the supernatant factor by studying the effects of interleukins (IL) 2-4, 6, and 8, granulocyte-macrophage colony stimulating factor, and tumor necrosis factor on the 35sulfate incorporation by rat glomeruli in vitro. A significant increase in GBM 35sulfate uptake was only seen when the glomeruli were cultured with the addition of IL-8 as compared with control cultures: 10.8 +/- (SEM) 1.7 and 7.9 +/- 1.4 cpm/micrograms GBM protein, respectively (p < 0.005). IL-8 reproduces the effect of the reported supernatant factor on the GBM 35sulfate uptake. Because IL-8 was detected in the supernatant of peripheral mononuclear cell cultures from idiopathic minimal-lesion nephrotic syndrome patients in relapse and because the increased GBM 35sulfate incorporation induced by the supernatant factor has been abolished by the addition to the culture media of anti-IL-8 neutralizing antibodies, we postulate that IL-8 is the previously described supernatant factor.

Transcriptional regulation of the tartrate-resistant acid phosphatase (TRAP) gene by iron

Tartrate-resistant acid phosphatase (TRAP) was first identified in cells from patients with hairy cell leukaemia. Subsequently, it has been found in other leukaemias, B-lymphoblastoid cell lines, osteoclasts and subsets of normal lymphocytes, macrophages, and granulocytes. Recent data indicate that TRAP and porcine uteroferrin, a placental iron-transport protein, represent a single gene product. However, the intracellular role of TRAP is unknown. We used a full-length human placental TRAP cDNA probe to examine TRAP expression in human peripheral mononuclear cells (PMCs). TRAP mRNA increased 50-75-fold after 24 h in unstimulated PMC cultures. Cell-fractionation experiments indicated that monocytes were the main cell population accounting for increased TRAP mRNA transcripts, and this was confirmed by histochemical staining for TRAP enzyme activity. Because expression of other iron-binding and -transport proteins is controlled by iron availability, we examined the role of iron in regulating TRAP expression. Increase of TRAP mRNA transcripts in PMCs was inhibited by 50 microM desferrioxamine, a potent iron chelator. The 5' flanking region of the TRAP gene was cloned from a mouse genomic library. In preliminary transient transfection experiments, it was determined that the 5'-flanking region of the TRAP gene contained iron-responsive elements. Therefore, a series of stably transfected HRE H9 cell lines was developed bearing genetic constructs containing various segments of the murine TRAP 5' promoter region driving a luciferase reporter gene. Treatment of transfectants with 100 micrograms/ml iron-saturated human transferrin (FeTF) was performed to assess iron responsiveness of the constructs. Constructs containing a full-length TRAP promoter (comprising base pairs -1846 to +2) responded to FeTF with a 4-5-fold increase of luciferase activity whereas constructs containing only base pairs -363 to +2 of the TRAP promoter did not respond. Constructs containing 1240 or 881 bp of the TRAP promoter gave only a 1.5- to 2-fold increase of luciferase activity with FeTF. In all cases, increase of luciferase activity was blocked by desferrioxamine. Cells transfected with another luciferase construct driven by a simian virus 40 promoter did not show any increase of luciferase activity with FeTF. These data indicate that expression of TRAP is regulated by iron and that this regulation is exerted at the level of gene transcription. The transfection experiments also suggest that the region of the TRAP 5'-flanking sequence between base pairs -1846 and -1240 contains an iron regulatory element.

Hexadecylphosphocholine-mediated enhancement of T-cell responses to interleukin 2

The effect of low-dose hexadecylphosphocholine (He-PC) on normal peripheral mononuclear cells (PMNC) was studied. Interferon-gamma (IFN-g) production, interleukin 2 (IL-2) receptor, and HLA-DR antigen expression were investigated, representing typical T-cell activation parameters. In PMNC cultures, He-PC dose-dependently enhanced the production of IFN-g, provided IL-2 had been added exogenously. Without IL-2 He-PC was ineffective. In some cultures, at a concentration of 8 μg/ml He-PC stimulated the secretion of IFN-g more than 20-fold compared to untreated controls. Although He-PC by itself lacked mitogenic activity, this compound also stimulated IFN-g production in the presence of suboptimal doses of phytohemagglutinin (PHA). Immunofluorescence studies demonstrated that He-PC also increased IL-2 receptor and HLA-DR antigen expression under these experimental conditions. Taken together, these results indicate that He-PC may possess immunomodulatory activity also in vivo, acting as a costimulator for the IL-2-mediated T-cell activation process.

In vitro IgE-secretion in atopic eczema: influence of allergens and mitogens and role of CD8 T cell subpopulation.

In cultures of peripheral blood mononuclear cells (PBMC) from 23 atopic patients and 14 controls the influence of mitogens, allergens and CD8 suppressor T lymphocytes on the in vitro IgE response was studied. The in vitro IgE levels in lymphocyte culture supernatants reached a plateau after 6 days of culture, whereby low levels of IgE could be reproducibly measured down to 0.5 ng/ml. The spontaneous in vitro IgE secretion from PBMC of atopic eczema patients was elevated in comparison to the control group and showed a direct correlation (r = 0.72) to the serum IgE levels. Pokeweed mitogen rather suppressed the in vitro IgE production. After removal of the CD8 subpopulation of T lymphocytes by using an indirect erythrocytes rosetting technique we found increased in vitro levels pointing to a role of IgE regulating T suppressor subpopulations.

Evaluation of two commercial tests for human immunodeficiency virus antigens in culture supernatant fluid.

Two commercial enzyme-linked immunosorbent assays (EIAs) for human immunodeficiency virus (HIV) antigens were evaluated for sensitivity and reproducibility by use of supernatant fluid from cell cultures of peripheral mononuclear cells of 18 patients with acquired immunodeficiency syndrome (AIDS) and 12 asymptomatic HIV antibody-positive patients. Both commercial assays detected HIV antigen in all cultures by day 21; however, the Abbott HIV antigen EIA (Abbott Laboratories, North Chicago, IL) detected HIV antigen three to seven days earlier in 15 of 48 cultures (31%), on the same day in 32 cultures (67%), and three days later in 1 culture (2%) when compared with the DuPont HIV antigen EIA (DuPont Laboratories, Wilmington, DE). In serial twofold dilution experiments, the Abbott HIV Ag EIA was found to have at least a twofold to eightfold increased sensitivity over the DuPont assay. Repeat testing of 15 initially positive supernatant fluids by both assays revealed that 13 of 15 and 12 of 15 were consistently positive by the Abbott and DuPont assays, respectively. The authors conclude that the Abbott EIA demonstrated better performance in this study than the DuPont EIA for detection of HIV in cell culture because of its shorter time-to-positivity and greater sensitivity.

Effects of natural human interferon-α, -β and -γ on interleukin 2 production in human peripheral lymphocytes

Effects of interferon (IFN) on PHA-induced interleukin 2 (IL-2) production by human peripheral mononuclear cells were studied comparatively with natural human IFN-α, IFN-β and IFN-γ, using an equivalent unit of their antiviral activity ranging from 10 to 1000 IU/ml. IL-2 activity was assessed in cultures with or without IFN by a standard bioassay using murine CTLL-2 cells. PHA-induced production of IL-2 in cultures of peripheral mononuclear cells was unaltered or slightly suppressed by the simultaneous presence of IFN-α and IFN-β. The effect was the same, whether or not indomethacin was present in the cultures. In contrast, the addition of IFN-γ to the PHA-stimulated cultures markedly enhanced IL-2 production, while IFN-γ per se had no effect on IL-2 production in the absence of PHA. The enhancement of IL-2 production due to IFN-γ was more marked in cultures which did not include indomethacin than in cultures which contained indomethacin (1×10 −6 M).

Humoral and cellular immune responsiveness to human S-antigen in uveitis.

Purified human retinal S-antigen (S-ag) was used to investigate the occurrence of humoral and cellular autoimmune reactions against S-ag in uveitis patients. With a sensitive ELISA method anti-S-ag antibodies could be detected in the sera of 28% of the uveitis patients. No difference was found between patients with posterior or panuveitis (31 out of 117 positive) and patients with anterior or intermediate uveitis (16 out of 52 positive). Similar frequencies and levels of anti-S-ag autoantibodies were also found among healthy controls (6/20) and patients who had undergone cataract surgery (6/17). Immunoblotting with purified S-ag and with whole human retinal extract confirmed the presence of anti-S-ag antibodies in uveitis and control sera. Moreover, antibodies against various other retinal proteins could also be demonstrated in patients and controls, without being particularly enhanced in uveitis. The cellular immune responsiveness was tested by measuring the production of migration inhibitory factor (MIF) during overnight culture of peripheral mononuclear cells with the antigen. None of 18 healthy controls responded, whereas 17 positive reactions were observed in the group of 44 uveitis patients. The highest frequencies were found in patients with posterior (5/12) or pan- (7/12) uveitis, while of the responders with anterior (2/8) or intermediate (3/12) uveitis, three had disorders affecting the retina. Thus, cellular autoimmune responsiveness to S-ag is apparently associated with posterior and pan-uveitis, and might also occur in non-uveitic retinal disorders, whereas the occurrence of anti-S-ag antibodies is probably not at all pathognomic for uveitis.

Interleukins in chronic active hepatitis B: Relationship with viral markers

Interleukin-2, a product of helper T cells, is essentially involved in the regulation of cell-mediated immunity. Two monocyte-derived factors, interleukin-1 and prostaglandin E2, influence interleukin-2 synthesis with opposite actions. To analyse immunoregulatory function in HBsAg-positive chronic active hepatitis, T cell subsets in peripheral blood and the levels of interleukin-2, interleukin-1 and prostaglandin E2 in supernatants from lectin- or lipopolysaccharide-activated peripheral mononuclear cell cultures were determined in 16 healthy controls and 33 patients with chronic active hepatitis B. Interleukin-2 activity was comparable to the controls in patients without delta infection who had seroconverted to anti-HBe (group 1), but it was significantly reduced in both HBeAg-positive subjects (group 2) (P less than 0.05 vs. controls and group 1) and those cases with positive delta markers (group 3) (P less than 0.01 and less than 0.05 vs. controls and group 1, respectively). In group 3, interleukin-2 was similarly diminished in both anti-HTLV-III-positive and -negative cases as well as in HBeAg- and anti-HBe-positive subjects. Notwithstanding the changes in interleukin-2 activity, no significant differences in the number of T4 cells, or in the levels of either interleukin-1 or prostaglandin E2, were found among the various groups of subjects studied. However, in those groups with reduced interleukin-2 activity an increased number of T8 cells was observed. It is suggested that the low levels of interleukin-2 found in the replicative phase of chronic active hepatitis B and in delta superinfection reflect a disturbed immunoregulation that may contribute to persistent viral replication in these two conditions.

Human lymphocytes synthesize C-reactive protein.

We obtained evidence for the synthesis and secretion of C-reactive protein (CRP) by peripheral mononuclear cells in culture. Human mononuclear cells isolated from peripheral blood, after depletion of platelets, were cultured in glutamine-depleted RPMI 1640 supplemented with [3H]glutamine in the presence of 10-O-tetradecanoyl-phorbol-13-acetate (TPA). Anti-CRP antiserum was added to the culture medium, and the resultant immunoprecipitate was analyzed in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The immunoprecipitate consisted of CRP, heavy and light chains of IgG, and only the CRP protein band had radioactivity, indicating that CRP was synthesized by mononuclear cells. In the populations of mononuclear cells, T-cell preparations mainly synthesized CRP, under stimulation of a factor derived from activated monocytes. Studies using the inhibitors of phospholipid metabolism suggested that generation of the monocyte factor was relevant to metabolites of an arachidonate cascade.

Effect of seasonal ragweed exposure on immunoglobulin E antiragweed antibodies in cultures of peripheral mononuclear cells, plasma and nasal secretions.

To determine the effects of seasonal ragweed exposure on in vitro IgE antibody production, the antiragweed IgE antibody content of 7-day cultures of peripheral blood mononuclear cells was assessed in samples obtained before, during and after the ragweed season, in 4 ragweed-allergic and 11 nonallergic individuals. The levels of ragweed-specific IgE and total IgE in unstimulated culture supernatants were measured by solid phase radioimmunoassays. Antiragweed IgE antibody was apparently detectable in the 7-day culture supernatants of both allergic and nonallergic individuals, and the antibody concentrations increased after natural ragweed exposure. However, the bindable counts of IgE antibody in supernatants from allergic patients were inhibited by soluble ragweed antigen, while those from nonallergics were not inhibitable, suggesting that supernatants from nonallergics did not contain true ragweed-specific IgE antibody. Because, for most subjects, the quantity of IgE in culture supernatants from 7-day cultures of living cells was the same as that derived from cells which were either frozen and thawed prior to culture, or briefly exposed to pH 3.7 buffer, the majority of IgE antibody in supernatants from allergic subjects, and of total IgE in supernatants from all subjects, was apparently not newly synthesized, but was preformed, and appeared to be largely derived from the cell surface. IgE antiragweed antibody was detected in the nasal secretions and plasma of allergic, but not nonallergic individuals, and the levels of antibody in allergic subjects increased after seasonal exposure. The fraction of total IgE protein accounted for by IgE antiragweed antibody in nasal secretions collected postseasonally was higher than that in both culture supernatants and plasma, suggesting that ragweed-specific IgE antibody is synthesized locally in the nose.