Traumatic brain injury and hemorrhage in a juvenile rat model of polytrauma leads to immunosuppression and splenic alterations.

BACKGROUND: Traumatic brain injury (TBI) is a common cause of morbidity and mortality. We have previously shown that TBI with a concurrent extra-cranial injury reliably leads to post-injury suppression of the innate immune system, but the impact of this injury on the adaptive immune system is unknown. We present data showing that combined injury reduced immune response as assayed in both blood and spleen samples and that these changes parallel apoptosis in the spleen. To assess the clinical relevance of these changes, we examined lungs for spontaneous bacterial colonization. METHODS: For these studies, prepubescent (28 day old) rats were injured using a controlled cortical impact model and then 25% blood volume removal by arteriotomy, and injured animals were compared with sham injured animals. Blood and spleen samples at post-injury day 1 were incubated with or without immunostimulant and examined for IFN-γ production using an Eli-Spot assay. Spleen samples were also examined for apoptosis using Annexin V staining, and lungs were harvested and plated on blood agar to examine for spontaneous bacterial colonization. RESULTS: Stimulations of whole blood and spleen samples with phorbol 12-myristate 13-acetate/ionomycin (PMA/I) at post-injury day 1 were associated with significant decreases in IFN-γ-positive cells/million in injured animals. Stimulation of whole blood with either PMA/I or pokeweed mitogen led to reduced tumor necrosis factor alpha production. Spleen from injured animals showed a marked increase in apoptosis. Lung samples showed a 300% increase in colonies per plate in injured animals. CONCLUSIONS: These data suggest that the combined injury can lead to adaptive immunosuppression, and our findings further suggest a potential role for the spleen in altering leukocyte function following injury.

Isolation of primary human B lymphocytes from tonsils compared to blood as alternative source for ex vivo application.

B lymphocytes ('B cells') are components of the human immune system with obvious potential for medical and biotechnological applications. Here, we discuss the isolation of primary human B cells from both juvenile and adult tonsillar material using a two-step procedure based on gradient centrifugation followed by separation on a nylon wool column as alternative to the current gold standard, i.e., negative immunosorting from buffy coats by antibody-coated magnetic beads. We show that the nylon wool separation is a low-cost method well suited to the isolation of large amounts of primary B cells reaching purities ≥ 80%. More importantly, this method allows the preservation of all B cell subsets, while MACS sorting seems to be biased against a certain B cell subtype, namely the CD27+ B cells. Importantly, compared to blood, the excellent recovery yield during purification of tonsillar B cells provides high number of cells, hence increases the number of subsequent experiments feasible with identical cell material, consequently improving comparability of results. The cultivability of the isolated B cells was demonstrated using pokeweed mitogen (PWM) as a stimulatory substance. Our results showed for the first time that the proliferative response of tonsillar B cells to mitogens declines with the age of the donor. Furthermore, we observed that PWM treatment stimulates the proliferation of a dedicated subpopulation and induces some terminal differentiation with ASCs signatures. Taken together this indicates that the proposed isolation procedure preserves the proliferative capability as well as the differentiation capacity of the B cells.

Early suppression of peripheral mononuclear blood cells in sepsis in response to stimulation with cytomegalovirus, OKT3, and pokeweed mitogen.

Critically ill patients are at risk for sepsis, and immunosuppressive mechanisms may prevail. Whether functional tests are helpful to detect immune alterations is largely unknown. Therefore, we tested the hypotheses that reactivity of peripheral blood mononuclear cells (PBMCs) to secrete interferon-γ (IFNγ) following stimulation in vitro is decreased in patients with early sepsis compared with postoperative patients. IFNγ secretion [enzyme-linked immunospot (ELISpot)] in response to stimulation with cytomegalovirus (CMV), pokeweed mitogen (PWM), muromonab-anti-CD3 (OKT3), and human leukocyte antigen (HLA)-DRA-mRNA expression and serum cytokine concentrations were repeatedly [days 1, 3, 5, and 7 after intensive care unit (ICU) admission] determined in patients with sepsis (n = 7) and patients undergoing major abdominal surgery (radical prostatectomy, cystectomy, n = 10). In a second cohort, HLA-DRA expression was assessed in 80 patients with sepsis, 30 postoperative patients, and 44 healthy volunteers (German clinical trials database no. 00007694). In patients with sepsis, IFNγ secretion (ELISpot) was decreased compared with controls after stimulation with CMV (P = 0.01), OKT3 (P = 0.02), and PWM (P = 0.02 on day 5), whereas unstimulated IFNγ secretion did not differ. HLA-DRA expression was also significantly decreased in patients with sepsis at all time points (P = 0.004) compared with postoperative surgical patients, a finding confirmed in the larger cohort. Reactivity of PBMCs to stimulation with CMV, PWM, and OKT3 as well as HLA-DRA expression was already decreased upon ICU admission in patients with sepsis when compared with postoperative controls, suggesting early depression of acquired immunity. ELISpot assays may help to clinically characterize the time course of immunocompetence in patients with sepsis.NEW & NOTEWORTHY We observed suppression of reactivity to stimulation with cytomegalovirus, muromonab-anti-CD3, and pokeweed mitogen in mononuclear blood cells of patients with early sepsis when compared with postoperative controls. Thus, there is early depression of acquired immunity in sepsis. Enzyme-linked immunospot assays may help to characterize immunocompetence in patients with sepsis.

Mitogen-Induced Interferon Gamma Production in Human Whole Blood: The Effect of Heat and Cations.

BACKGROUND: Interferon-gamma release assays (IGRAs) are blood tests used to measure the amount of interferon-γ (IFN-γ) released by T lymphocytes after stimulation by antigens specific for the diagnosis of latent tuberculosis infection. A mitogen serves as a positive control to assess the immune function in IGRAs. METHODS: This in vitro study was conducted to evaluate IFN-γ production by human whole blood stimulated with heat-treated and/or cation-supplemented phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM), using QuantiFERON-TB Gold Kit ELISA tests. RESULTS: The optimal concentrations of PWM, Con A and PHA for IGRAs were 2 µg/mL, 5 µg/mL and 10 µg/mL, respectively. The results showed that IFN-γ production in response to PWM was the highest and PHA was the lowest amount. The median values of three mitogens were in the following order: PWM≥Con A≥ positive control>>PHA-P>>negative control. PWM and PHA were heat stable, while Con A was heat sensitive. The mitogen response of lymphocytes to untreated or heat-treated PWM and heat-treated Con A was increased in 1 mM Ca2+-supplemented groups, whereas the response to heat-treated PHA was decreased. Exposure to 1 mM Mg2+ had no effect on untreated or heat-treated PWM, and a concentration of 1 mM Zn2+ inhibited the stimulation of un-treated PWM. We found that calcium supplementation improved the PWM-induced production of IFN-γ. CONCLUSION: Therefore, PWM is an appropriate mitogen for use as a positive control in IGRAs. It is a potential indicator of cytokine production in the diagnostic as well as research settings, and calcium supplementation improved stimulation.

Immunomodulatory and cellular antioxidant activities of pure compounds from Teucrium ramosissimum Desf.

Evaluation of the immunomodulatory activity of plant compounds is an interesting and growing area of research. Teucrium ramosissimum Desf. is a native and endemic medicinal plant from the South of Tunisia traditionally used for the treatment of many diseases. The anti-inflammatory activity of apigenin-7-glucoside, genkwanin, and naringenin isolated from T. ramosissimum were assayed. The phagocytic activities of macrophage and lymphocyte proliferation were investigated in the absence and presence of mitogens (lipopolysaccharide [LPS] or lectin). Depending on the concentrations, the compounds affect macrophage functions by modulating their lysosomal enzyme activity and nitric oxide (NO) release. The tested compounds enhance significantly splenocyte proliferation, either with or without mitogen stimulation. In studies to assess any potential effects of apigenin-7-glucoside, genkwanin, and naringenin on innate immunity, the results showed that these compounds significantly enhanced the killing activity of natural killer (NK) cells and cytotoxic activity of the T lymphocyte (CTL) isolated from splenocytes. These results suggest that T. ramosissimum compounds such as apigenin-7-glucoside, genkwanin, and naringenin may be potentially useful for modulating immune cell functions in physiological and pathological conditions.

Defective B-cell proliferation and maintenance of long-term memory in patients with chronic granulomatous disease.

BACKGROUND: Chronic granulomatous disease (CGD) is a primary immune deficiency characterized by a defect in reactive oxygen species production. Although the effect of CGD mainly reflects on the phagocytic compartment, B-cell responses are also impaired in patients with CGD. OBJECTIVE: We sought to investigate how defective gp91(phox) expression in patients with CGD and CGD carriers might affect the B-cell compartment and maintenance of long-term memory. METHODS: We studied the B-cell compartment of patients with CGD in terms of phenotype and ability to produce reactive oxygen species and proliferate on stimuli differently directed to the B-cell receptor and Toll-like receptor 9. We further studied their capacity to maintain long-term memory by measuring cellular and serologic responses to measles. RESULTS: We show that the memory B-cell compartment is impaired among patients with CGD, as indicated by reduced total (CD19(+)CD27(+)) and resting (CD19(+)CD27(+)CD21(+)) memory B cells in parallel to increased naive (CD19(+)CD27(-)IgD(+)) B-cell frequencies. Data on CGD carriers reveal that such alterations are related to gp91(phox) expression. Moreover, proliferative capabilities of B cells on selective in vitro stimulation of B-cell receptor or Toll-like receptor 9 pathways were reduced in patients with CGD compared with those seen in age-matched healthy control subjects. Significantly lower measles-specific antibody levels and antibody-secreting cell numbers were also observed, indicating a poor ability to maintain long-term memory in these patients. CONCLUSION: Altogether, our data suggest that patients with CGD present a defective B-cell compartment in terms of frequencies of memory B cells, response to in vitro stimulation, and maintenance of long-term antigen-specific memory.

Raman spectroscopic modeling of early versus late T-lymphocyte activation via differential spectral detection of receptor expression.

The proven efficacy of renal transplantation has made it the definitive treatment for end-stage renal disease. Despite its wide acceptance, transplantation has been limited by organ shortages. In the face of this, preservation of allograft longevity is essential. The predominately T cell-driven process of acute rejection (AR) can lead to graft dysfunction and even graft loss. As a marker for AR screening, serum creatinine has a low sensitivity and specificity. This has warranted the development of more accurate screening/diagnostic tools such as Raman Spectroscopy (RS) which has been demonstrated in previous studies to accurately quantify T cell activation. In this study we further explore its application by modeling the dynamic process of cell surface receptor expression during T cell activation. 50 mitogen (Concanavalin A and pokeweed) activated T cells were stained with CD69, CD25, and CD71 monoclonal antibodies (mAbs) at 48 and 72 hour time points. In parallel, 50 activated T cells were analyzed using RS at these same time periods. At 4 8h there was high expression of the CD69 cell surface receptor detected via mAb staining with no appreciable binding of CD25/CD71 fluorescent tag. In conjunction, 48 hour RS-analyzed T cells demonstrated a significant peak difference at the 1585 cm(-1) position which represented a 63% (p=0.01) increase in peak magnitude when compared with the 72 hour samples. By contrast, the 72 hour data demonstrated an attenuation of the CD69 expression and increased CD25/CD71 expression. The corresponding RS analysis showed two significant peak differences at the 903 cm(-1) and 1449 cm(-1) positions that were not present at 48 h. These differences in Raman shifts resulted in a 40% (p=0.04) and a 59% (p=0.001) increase in peak magnitudes at these positions, respectively. This study serves to further validate RS as a screening modality capable of not only detecting T cells early in the activation process through the spectral signatures associated with CD69, but also quantifying the persistent expression of CD25 and CD71. This provides a foundation for the development of a system capable of the accurate assessment of acute and maintenance immunosuppression efficacy at the molecular level.

Evaluation of T and B lymphocyte function in clinical practice using a flow cytometry based proliferation assay.

The golden standard for functional evaluation of immunodeficiencies is the incorporation of [(3)H]-thymidine in a proliferation assay stimulated with mitogens. Recently developed whole blood proliferation assays have the advantage of parallel lymphocyte lineage analysis and in addition provide a non-radioactive alternative. Here we evaluate the Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) in a comparison with [(3)H]-thymidine incorporation in four patients with severe combined immunodeficiency. The threshold for the minimum number of lymphocytes required for reliable responses in FASCIA is determined together with reference values from 100 healthy donors when stimulated with mitogens as well as antigen specific stimuli. Finally, responses against PWM and SEA+SEB stimuli are conducted with clinically relevant immunomodulatory compounds. We conclude that FASCIA is a rapid, stable and sensitive functional whole blood assay that requires small amounts of whole blood that can be used for reliable assessment of lymphocyte reactivity in patients.

Leukocyte proliferation and immune modulator production in patients with chronic kidney disease.

INTRODUCTION: In Chronic Kidney Disease (CKD), immune cells are affected by uremic retention toxins. Given this effect, we analyzed lymphocyte proliferative response and immune modulators production following in vitro stimulation. METHODS: Whole blood was drawn from healthy controls, patients with eGFR <20 ml/min/1.73 m(2) (Pre-dialysis, CKD stages 4 and 5) and hemodialysis patients (stage 5D). Peripheral cells were incubated for six days with pokeweed mitogen, concanavalin A, Staphylococcus enterotoxin A or influenza A vaccine. Peripheral lymphocyte proliferation was then analyzed by the "Flow-cytometric Assay of Specific Cell-mediated Immune response in Activated whole blood" (FASCIA) method, and cytokine profile in the cell supernatants was analyzed by the Milliplex multi-array method. RESULTS: The absolute number of lymphoblasts in response to mitogenic stimulation and the number of cells in each CD4+ and CD8+ subpopulation were similar comparing the three groups, except for a single decline in number of lymphoblasts after stimulation with Staphylococcus enterotoxin A, comparing dialysis patients with healthy controls. Levels of interleukin (IL)-2 (p=0.026), -10 (p=0.019) and -15 (p=0.027) in the Staphylococcus enterotoxin A-stimulated supernatant were lower in hemodialysis patients compared to healthy controls. Levels of IL-15 (p=0.017) from pre-dialysis patients and levels of IL-5 (p=0.019) from hemodialysis patients in influenza A vaccine-stimulated supernatants were also lower compared to controls. In pokeweed mitogen-stimulated supernatant, IL-2 levels (p=0.013) were lower in hemodialysis patients compared to pre-dialysis patients. TNF-α, IL-10, IL-12, IL-15, IL-8, MCP-1, IP-10, IFN-α2, IL-1α and eotaxin levels were all significantly higher in plasma obtained from CKD patients. CONCLUSION: Our results suggest that T-cells from CKD patients have similar proliferative response to stimulation compared with healthy individuals. Moreover, however the immune cells show inability to produce selected cytokines, most likely due to the uremic milieu or dialysis procedure.

A novel anti-human ICOSL monoclonal antibody that enhances IgG production of B cells.

ICOSL, a newly identified member of the B7 superfamily, plays a major role in immune responses. In this study, a functional anti-human ICOSL monoclonal antibody (MAb) 3B3 was obtained and characterized by means of flow cytometry, Western blot, and competition assay. This MAb could specifically recognize a distinct epitope of the ICOSL molecule. As a functional antibody, MAb 3B3 could inhibit the proliferation of T lymphocytes stimulated by ICOSL-L929 transfectants. Furthermore, it could enhance IgG production of PWM-driven B cells. The results indicate that the ICOS-ICOSL signal is critically involved in specific humoral immunity.

Proliferative response of lymphocyte to pokeweed mitogen depends on the concentration of endogenous cortisol in the early post-traumatic period in patients with penetrating eye injury.

The intensity of lymphocyte proliferation in response to pokeweed mitogen depends on cortisol level in the peripheral blood in the early post-traumatic period of penetrating eye injury. Lymphocyte proliferation in 72- and 96-h cultures from patients with high levels of endogenous hormone was suppressed. In 120-h cultures, the intensity of proliferation remains unchanged. Lymphocyte blast transformation was increased in 120-h cultures from patients with normal cortisol concentration and remained unchanged in case of low cortisol level.

Poke weed mitogen requires Toll-like receptor ligands for proliferative activity in human and murine B lymphocytes.

Poke weed mitogen (PWM), a lectin purified from Phytolacca americana is frequently used as a B cell-specific stimulus to trigger proliferation and immunoglobulin secretion. In the present study we investigated the mechanisms underlying the B cell stimulatory capacity of PWM. Strikingly, we observed that highly purified PWM preparations failed to induce B cell proliferation. By contrast, commercially available PWM preparations with B cell activity contained Toll-like receptor (TLR) ligands such as TLR2-active lipoproteins, lipopolysaccharide and DNA of bacterial origin. We show that these microbial substances contribute to the stimulatory activity of PWM. Additional experimental data highlight the capacity of PWM to enable B cell activation by immunostimulatory DNA. Based on these findings we propose that the lectin sensitizes B cells for TLR stimulation as described for B cell receptor ligation and that B cell mitogenicity of PWM preparations results from synergistic activity of the poke weed lectin and microbial TLR ligands present in the PWM preparations.

Lenalidomide alone or lenalidomide plus dexamethasone significantly inhibit IgG and IgM in vitro... A possible explanation for their mechanism of action in treating multiple myeloma.

Lenalidomide (len) is an analog of thalidomide (thal), and both are used in the treatment of a diverse group of medical conditions. A common finding in this group is the detection of immunoglobulin in skin lesions, or high levels of immunoglobulin or myeloma protein in serum and urine. While their mechanism(s) of action is not known, the drugs are noted for their ability to modulate monocyte, lymphocyte, and natural killer cell functions; suppression of immunoglobulin synthesis could offer an explanation for their effectiveness in treating multiple myeloma (MM). Our objective was to determine if, on an equimolar basis, thal, len or dexamethasone (dex) could affect pokeweed (PWM)-induced synthesis of IgG, IgM and IL-2. When peripheral blood mononuclear cells were stimulated with PWM, len surpassed thal in suppressing IgM and IgG, and enhancing IL-2. Dex enhanced IgG, and suppressed IL-2. When the stimulated cells were treated with len (an effective promoter of IL-2 and suppressor of IgM and IgG) plus dex (an effective suppressor of IL-2 and enhancer of IgG), the net result was suppression of IgM and IgG. The synthesis of IgM and IgG by putative PWM-stimulated B cell blasts is significantly blocked by len. This suggest that the B-lymphocyte is a targeted cell for len, and that suppression of the synthesis of IgG and IgM could provide an explanation for the mechanism by which len effectively treats MM.

Induced cytokine response of human PMBC-cultures: correlation of gene expression and secretion profiling and the effect of cryopreservation.

The immune system is regulated by the complex interaction of multiple cytokines, which are secreted signaling molecules affecting other cells. In this work, we studied the cytokine response to several well-known stimulants, such as OKT-3, Con A, PWM, and SEB. Healthy donor cells (PBMCs) were cultivated for up to 72 h and the mRNA levels and cytokine release of four key cytokines (IL-2, IL-4, IFN-γ, and TNF-α) were analyzed by RT-PCR and bead-based multiplex analyses. The generated cytokine profiles showed characteristic expression patterns and secretion kinetics for each cytokine and substance. PWM/SEB and OKT-3 led to a very fast and long-lasting immune response, whereas Con A induced the slowest cytokine production. Cytokine concentrations also differed greatly. The highest IFN-γ concentration was 1000 times higher than the respective IL-4 concentration. Gene expression and cytokine concentration profiles were strongly correlated during the time course. The chronological response of the donors' cytokine profiles coincided, but showed individual characteristics regarding the strength of the cytokine release. The comparison of stimulation experiments using freshly isolated and cryopreserved PBMCs showed that, for the observation of an immunological response at early points in time, gene expression experiments are more reliable than the measurement of cytokines in the cell culture supernatant. However, the freezing of cells influences the response significantly. The measurement of secreted proteins is the superior method at later points in time.

Human mesenchymal stem cell co-culture modulates the immunological properties of human intervertebral disc tissue fragments in vitro.

The capacity of mesenchymal stem cells (MSCs) to differentiate into intervertebral disc (IVD)-like cells has been well described, but their ability to modulate the inflammatory processes in the IVD remains unclear. We found that tissue obtained by discectomy of degenerated and post-traumatic IVD contains significant amounts of IgG antibodies, a sign of lymphocyte infiltration. Further we investigated whether MSCs in vitro, which were characterized for their multilineage differentiation potential and may have immunomodulatory effects on IVD fragments. IVD fragments were co-cultured in contact with peripheral blood lymphocytes (PBLs) and MSCs, and as functional controls we used contact co-cultures of PBLs stimulated with pokeweed mitogen (2.5 µg/mL) and MSCs. The time course of lymphocyte proliferation (Alamar Blue), IgG (ELISA) and gene expression (RT-PCR) of anti-inflammatory cytokines (TGF-ß1, IL-10) by MSCs and pro-inflammatory molecules (IL-1α, IL-1ß and TNF-α) by the IVD fragments were analyzed. Depending on the response to the presence of MSCs, the IVD fragments (n = 13) were divided in two groups: responders (n = 9), where inflammation was inhibited by MSCs and non-responders (n = 4), where MSCs did not decrease inflammation. At 1 week in co-culture, MSCs reduced significantly the IgG production in the IVD responders group to 69% and PBLs proliferation to 57% of the control. MSCs expression of the anti-inflammatory TGF-ß1 increased with time, while IL-10 was expressed only at day 1. IVD gene expression of TNF-α decreased constantly, whereas IL-1α and IL-1ß expression increased. In conclusion, these data suggest that MSCs may modulate disc-specific inflammatory and pain status and aid regeneration of the host tissue.

Thalidomide inhibited the synthesis of IgM and IgG whereas Thalidomide+Dexamethasone and Dexamethasone alone acted as co-stimulants with pokeweed and enhanced their synthesis.

Thalidomide (Thal) provides effective treatment for erythema nodosum leprosum (ENL). In combination with Dexamethasome (Dex) it is an effective treatment for multiple myeloma (MM) and Waldenström's macroglobulinemia (WM). Thal's mechanism(s) of action in the treatment of these diverse medical conditions is not known, but it could be suppression of immunoglobulin (Ig) synthesis. Mononuclear cells were stimulated with pokeweed (PWM), and treated with Thal, Thal+Dex or Dex. The cultures were assayed for IgM and IgG. The maximum synthesis was expected to occur in cultures stimulated with PWM at 0.5, 5.0 or 10 microg/ml. The test agents at 15 microM each were expected to alter the response. Compared to cultures stimulated with PWM alone, there was significantly less Ig in the cultures containing Thal+PWM, and significantly more Ig in the cultures containing Thal+Dex+PWM or Dex+PWM (Wilcoxon). The median % of maximum was 57 for cultures treated with Thal+PWM; 184 for cultures treated with Thal+Dex+PWM, and 139 for cultures treated with Dex+PWM. Thal also acted as a co-stimulant with PWM and enhanced the synthesis of IL-2, IL-6 and DNA; whereas, Thal+Dex or Dex enhanced Ig synthesis, but suppressed IL-2, IL-6 and cell proliferation. Thal's ability to suppress Ig may explain its activity in ENL, MM and WM. The enhancement of Ig by Dex does not help to explain a role for Dex alone or in combination with Thal for the treatment of MM and WM.

Improvement of in vitro oocyte maturation with lectin supplementation and expression analysis of Cx43, GDF-9, FGF-4 and Fibronectin mRNA transcripts in Buffalo (Bubalus bubalis).

PURPOSE: To optimize In vitro maturation (IVM) of quality oocytes for embryo production through IVF and SCNT. METHODS: Buffalo oocytes were in vitro matured in the presence of the pokeweed lectin (Phytolacca americana), a potent lymphocyte mitogen. Lectin was supplemented in TCM + 10% FBS at the doses of 0, 1, 5, 10, 15, 20 and 40 microg/ml and cumulus expansion and gene expression patterns were characterized. RESULTS: The degree of cumulus expansion in different lectin treatment levels improved from 1.1 at 1 Ag/ml level to 3.60 at 10 microg/ml level and then decreased in higher concentration 20 microg/ml (1.66) and 40 microg/ml (0.64). IVF embryos showed highest cleavage rate (88.8%) in 10 microg/ml lectin treatment. Expression of all mRNA transcript studied (Cx43, GDF 9, FGF-4 and Fibronectin) was positively correlated with cumulus expansion and polar body extrusion. CONCLUSIONS: Mitogenic lectin supplemented maturation media improves oocyte quality for in vitro embryo production.

Increased spontaneous but decreased mitogen-stimulated expression and excretion of interleukin 18 by mononuclear cells in patients with active systemic lupus erythematosus.

OBJECTIVE: To measure serum concentration and analyze the expression of interleukin 18 (IL-18) mRNA in mononuclear cells of patients with systemic lupus erythematosus (SLE). METHODS: IL-18 concentrations in sera and culture supernatants of peripheral blood mononuclear cells (PBMC) from healthy controls and patients with active SLE were measured by ELISA. PBMC and polymorphonuclear leukocytes (PMN) purified from patients with active SLE were stimulated with phytohemagglutinin (PHA), pokeweed mitogen (PWM), and lipopolysaccharide (LPS). Expression of IL-18 mRNA in cells was analyzed by RT-PCR. RESULTS: Serum IL-18 levels were significantly higher in SLE patients than in controls, and correlated with disease activity in SLE patients (r(2) = 0.602). Two patients receiving intravenous methylprednisolone therapy (1.0 g/day for 3 days) showed profound decreases in serum IL-18 levels after therapy. The quiescent PBMC from SLE patients (30/30) expressed IL-18 transcript more frequently than control PBMC (20/30). PBMC from SLE patients produced more IL-18 than control PBMC after 72 hours of incubation, by RT-PCR. PHA and PWM inhibited the production of IL-18 in PBMC from both SLE patients and controls. Inhibition by PWM was more pronounced than that by PHA, especially in SLE-PBMC. Control and SLE-PMN with or without LPS stimulation produced negligible IL-18. CONCLUSION: IL-18 is involved in the autoimmune derangement of leukocyte function in patients with active SLE.

Effects of diabetes mellitus vs. in vitro hyperglycemia on select immune cell functions.

Diabetes mellitus (DM), one of the commonest metabolic disorders, can impair the function of cells involved in cellular and/or humoral immunity. This study sought to define potential effects upon cell-mediated immune cells due to an acute hyperglycemic state (in vitro) for comparison against those that might be attributable to a diabetic phenotype itself. Peripheral blood mononuclear cells (PBMC) were isolated from ten diabetic patients (5 with Type I disease and 5 with Type II) and 10 healthy controls. The cells were then challenged with 1 of 3 different mitogens (concanavalin A, phytohemagglutinin, pokeweed mitogen) in the presence of differing glucose concentrations (0, 100, 200, 400, or 800 mg/dl), and proliferative responses assessed. Neutrophils (PMNC) from the blood samples, exposed to the same experimental conditions, were analyzed for respiratory burst activity using nitroblue tetrazolium. The results indicated that there was significant inhibition of the proliferative responses to mitogens among the stimulated PBMC and in respiratory burst activity among the PMNC obtained from the diabetic patients. However, these effects were not affected by either the added presence of increasing amounts of exogenous glucose, the type of diabetes the patients had, the length of time the patient had had the disease, or whether or not the patients had been receiving insulin treatments. In contrast, the PBMC from healthy individuals appeared to display dose-trend decreases in responsiveness to mitogens; interestingly, similar effects on their PMNC were not evident. It was thus concluded that in situ ongoing repeated hyperglycemic states caused changes in cells of the immune system that could have been caused by repeated "continuous" exposures to excess sugar. Further studies are needed to more clearly identify hyperglycemia (sugar)-sensitive targets on/in these cells that could contribute to the appearance of the diabetic immunodeficiency in these types of patients.

Atopic dermatitis in adults: evaluation of peripheral blood mononuclear cells proliferation response to Staphylococcus aureus enterotoxins A and B and analysis of interleukin-18 secretion.

BACKGROUND: Atopic dermatitis (AD) is a chronic, inflammatory skin disease with a high prevalence and complex pathogenesis. The skin of AD patients is usually colonized by Staphylococcus aureus (S. aureus); its exotoxins may trigger or enhance the cutaneous inflammation. Several mediators are related to the AD immune imbalance and interleukin-18 (IL-18), an inflammatory cytokine, may play a role in the atopic skin inflammation. AIMS: To evaluate peripheral blood mononuclear cells (PBMC) proliferation response to staphylococcal enterotoxins A (SEA) and B (SEB) and the levels of IL-18 in adults with AD. METHODS: Thirty-eight adult patients with AD and 33 healthy controls were analysed. PBMC were stimulated with SEA and SEB, phytohemaglutinin (PHA), pokeweed (PWM), tetanus toxoid (TT) and Candida albicans (CMA). IL-18 secretion from PBMC culture supernatants and sera were measured by ELISA. RESULTS: A significant inhibition of the PBMC proliferation response to SEA, PHA, TT and CMA of AD patients was detected (P < or = 0.05). Furthermore, increased levels of IL-18 were detected both in sera and non-stimulated PBMC culture supernatants from AD patients (P < or = 0.05). CONCLUSIONS: A decreased PBMC proliferation response to distinct antigens and mitogens (TT, CMA, SEA and PHA) in adults with AD suggest a compromised immune profile. IL-18 secretion from AD upon stimulation was similar from controls, which may indicate a diverse mechanism of skin inflammation maintained by Staphylococcus aureus. On the other hand, augmented IL-18 secretion from AD sera and non-stimulated cell culture may enhance the immune dysfunction observed in AD, leading to constant skin inflammation.