Immune profiling in multiple sclerosis: a single-center study of 65 cytokines, chemokines, and related molecules in cerebrospinal fluid and serum.

Introduction: The understanding of the pathophysiology of multiple sclerosis (MS) has evolved alongside the characterization of cytokines and chemokines in cerebrospinal fluid (CSF) and serum. However, the complex interplay of pro- and anti-inflammatory cytokines and chemokines in different body fluids in people with MS (pwMS) and their association with disease progression is still not well understood and needs further investigation. Therefore, the aim of this study was to profile a total of 65 cytokines, chemokines, and related molecules in paired serum and CSF samples of pwMS at disease onset. Methods: Multiplex bead-based assays were performed and baseline routine laboratory diagnostics, magnetic resonance imaging (MRI), and clinical characteristics were assessed. Of 44 participants included, 40 had a relapsing-remitting disease course and four a primary progressive MS. Results: There were 29 cytokines and chemokines that were significantly higher in CSF and 15 in serum. Statistically significant associations with moderate effect sizes were found for 34 of 65 analytes with sex, age, CSF, and MRI parameters and disease progression. Discussion: In conclusion, this study provides data on the distribution of 65 different cytokines, chemokines, and related molecules in CSF and serum in newly diagnosed pwMS.

Multiple sclerosis-disease modifying therapies affect humoral and T-cell response to mRNA COVID-19 vaccine.

Background: The mRNA vaccines help protect from COVID-19 severity, however multiple sclerosis (MS) disease modifying therapies (DMTs) might affect the development of humoral and T-cell specific response to vaccination. Methods: The aim of the study was to evaluate humoral and specific T-cell response, as well as B-cell activation and survival factors, in people with MS (pwMS) under DMTs before (T0) and after two months (T1) from the third dose of vaccine, comparing the obtained findings to healthy donors (HD). All possible combinations of intracellular IFNγ, IL2 and TNFα T-cell production were evaluated, and T-cells were labelled "responding T-cells", those cells that produced at least one of the three cytokines of interest, and "triple positive T-cells", those cells that produced simultaneously all the three cytokines. Results: The cross-sectional evaluation showed no significant differences in anti-S antibody titers between pwMS and HD at both time-points. In pwMS, lower percentages of responding T-cells at T0 (CD4: p=0.0165; CD8: p=0.0022) and triple positive T-cells at both time-points compared to HD were observed (at T0, CD4: p=0.0007 and CD8: p=0.0703; at T1, CD4: p=0.0422 and CD8: p=0.0535). At T0, pwMS showed higher plasma levels of APRIL, BAFF and CD40L compared to HD (p<0.0001, p<0.0001 and p<0.0001, respectively) and at T1, plasma levels of BAFF were still higher in pwMS compared to HD (p=0.0022).According to DMTs, at both T0 and T1, lower anti-S antibody titers in the depleting/sequestering-out compared to the enriching-in pwMS subgroup were found (p=0.0410 and p=0.0047, respectively) as well as lower percentages of responding CD4+ T-cells (CD4: p=0.0394 and p=0.0004, respectively). Moreover, the depleting/sequestering-out subgroup showed higher percentages of IFNγ-IL2-TNFα+ T-cells at both time-points, compared to the enriching-in subgroup in which a more heterogeneous cytokine profile was observed (at T0 CD4: p=0.0187; at T0 and T1 CD8: p =0.0007 and p =0.0077, respectively). Conclusion: In pwMS, humoral and T-cell response to vaccination seems to be influenced by the different DMTs. pwMS under depleting/sequestering-out treatment can mount cellular responses even in the presence of a low positive humoral response, although the cellular response seems qualitatively inferior compared to HD. An understanding of T-cell quality dynamic is needed to determine the best vaccination strategy and in general the capability of immune response in pwMS under different DMT.

SARS-CoV-2 pneumonia and bacterial pneumonia patients differ in a second hit immune response model.

Secondary infections have been shown to complicate the clinical course and worsen the outcome of critically ill patients. Severe Coronavirus Disease 2019 (COVID-19) may be accompanied by a pronounced cytokine release, and immune competence of these patients towards most pathogenic antigens remains uncompromised early in the disease. Patients with bacterial sepsis also exhibit excessive cytokine release with systemic hyper-inflammation, however, typically followed by an anti-inflammatory phase, causing immune paralysis. In a second hit immune response model, leukocyte activation capacity of severely ill patients with pneumonia caused by SARS-CoV-2 or by bacteria were compared upon ICU admission and at days 4 and 7 of the ICU stay. Blood cell count and release of the pro-inflammatory cytokines IL-2, IFNγ and TNF were assessed after whole-blood incubation with the potent immune stimulus pokeweed mitogen (PWM). For comparison, patients with bacterial sepsis not originating from pneumonia, and healthy volunteers were included. Lymphopenia and granulocytosis were less pronounced in COVID-19 patients compared to bacterial sepsis patients. After PWM stimulation, COVID-19 patients showed a reduced release of IFNγ, while IL-2 levels were found similar and TNF levels were increased compared to healthy controls. Interestingly, concentrations of all three cytokines were significantly higher in samples from COVID-19 patients compared to samples from patients with bacterial infection. This fundamental difference in immune competence during a second hit between COVID-19 and sepsis patients may have implications for the selection of immune suppressive or enhancing therapies in personalized medicine.

A ferret model of immunosuppression induced with dexamethasone.

Ferrets are nowadays frequently used as animal models for biomedical purposes; in many cases, immunosuppression of experimental animals is necessary. The aim of this study was to evaluate the effect of intramuscular dexamethasone administration (2 mg/kg as the initiation dose continued with 1 mg/kg q 12 h applied 5 times) on ferret's immune system. In comparison with ferrets which received the saline (n = 5), significantly lower total counts of leukocytes (P < 0.01), lymphocytes (P < 0.01) and monocyte (P < 0.05), as well as absolute numbers of CD4+CD8- (P < 0.01) and CD4-CD8+ (P < 0.01) subsets were noted in dexamethasone treated ferrets (n = 5) the first day after the treatment (D1). Absolute number of CD79+ lymphocytes remained unchanged throughout the experiment. The proliferation activity of lymphocytes in dexamethasone treated ferrets was lower only in D1 using concanavalin A (conA), phytohemagglutinin (PHA) and pokeweed mitogen (PWM); statistical significance was noted using PHA 40 (P < 0.05) and PWM 10 (P < 0.01). Lower neutrophil activity (P < 0.01) was detected in D1 after the dexamethasone treatment in both production of reactive oxygen species (chemiluminescence test) and ingestion of particles (phagocytosis assay). The dexamethasone treatment proved to be useful for short-term immunosuppression in ferrets. The results closely resembled data previously reported in human studies and indicate classification of ferrets as steroid-resistant species.

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.

Proliferation of peripheral blood mononuclear cells from healthy piglets after mitogen stimulation as indicators of disease resilience.

Disease resilience refers to the productivity of an animal under disease. Given the high biosecurity of pig nucleus herds, traits that can be measured on healthy pigs and that are genetically correlated with disease resilience, that is, genetic indicator traits, offer a strategy to select for disease resilience. Our objective was to evaluate mitogen stimulation assays (MSAs) on peripheral blood mononuclear cells (PBMCs) from young healthy pigs as genetic indicators for disease resilience. Data were from a natural disease challenge in which batches of 60 or 75 naïve Yorkshire × Landrace piglets were introduced every 3 wk into a continuous flow barn that was seeded with multiple diseases. In this environment, disease resilience traits, including growth, treatment, and mortality rates, were recorded on 3,136 pigs that were genotyped with a high-density marker panel. PBMCs from 882 of these pigs from 19 batches were isolated from whole blood collected prior to the disease challenge and stimulated with five mitogens: concanavalin A (ConA), phytohemagglutinin (PHA), pokeweed mitogen (PWM), lipopolysaccharide (LPS), and phorbol myristate acetate (PMA). The proliferation of cells was evaluated at 48, 72, and 96 h and compared with unstimulated samples (rest count). Heritabilities of cell proliferation were estimated using a model with batch as a fixed effect and covariates of entry age; rest count; complete blood count proportions of lymphocytes, monocytes, eosinophils, and basophils; and pen, litter, and animal genetics as random effects. Heritability estimates were highest for response to ConA (0.30 ± 0.09, 0.28 ± 0.10, 0.17 ± 0.10, and 0.25 ±0.10 at 48, 72, and 96 h after stimulation and for area under the curve across the three time points, respectively). Estimates were in a similar range for response to PHA and PMA but low for PWM and LPS. Responses to ConA, PHA, and PMA were moderately genetically correlated with several disease resilience traits and in the expected direction, but individual estimates were not significantly different from zero due to large SEs. In conclusion, although validation is needed, MSAss, in particular based on ConA, show promise as genetic indicator traits for disease resilience.

CellTrace Violet™ inhibits equine lymphocyte proliferation.

CellTrace Violet™ is a commonly used fluorescent dye used with flow cytometry to identify cell proliferation. Activated equine lymphocytes were examined using flow cytometry, microscopy and tritiated thymidine proliferation assays. CellTrace Violet™ was incorporated into the equine lymphocytes effectively. Equine lymphocytes proliferated when activated with pokeweed mitogen, but did not proliferate when previously stained with CellTrace Violet™. Serial dilutions of CellTrace Violet™ did not eliminate the inhibition of activated lymphocytes. Equine lymphocyte viability was greater than 90 % for both stained and unstained cells. Based on these data, CellTrace Violet™ is not recommended for the assessment of lymphocyte proliferation in equine cells. The mechanism of inhibition of equine lymphocyte proliferation by CellTrace Violet™ is unknown.

Quinolinate as a Marker for Kynurenine Metabolite Formation and the Unresolved Question of NAD+ Synthesis During Inflammation and Infection.

Quinolinate (Quin) is a classic example of a biochemical double-edged sword, acting as both essential metabolite and potent neurotoxin. Quin is an important metabolite in the kynurenine pathway of tryptophan catabolism leading to the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). As a precursor for NAD+, Quin can direct a portion of tryptophan catabolism toward replenishing cellular NAD+ levels in response to inflammation and infection. Intracellular Quin levels increase dramatically in response to immune stimulation [e.g., lipopolysaccharide (LPS) or pokeweed mitogen (PWM)] in macrophages, microglia, dendritic cells, and other cells of the immune system. NAD+ serves numerous functions including energy production, the poly ADP ribose polymerization (PARP) reaction involved in DNA repair, and the activity of various enzymes such as the NAD+-dependent deacetylases known as sirtuins. We used highly specific antibodies to protein-coupled Quin to delineate cells that accumulate Quin as a key aspect of the response to immune stimulation and infection. Here, we describe Quin staining in the brain, spleen, and liver after LPS administration to the brain or systemic PWM administration. Quin expression was strong in immune cells in the periphery after both treatments, whereas very limited Quin expression was observed in the brain even after direct LPS injection. Immunoreactive cells exhibited diverse morphology ranging from foam cells to cells with membrane extensions related to cell motility. We also examined protein expression changes in the spleen after kynurenine administration. Acute (8 h) and prolonged (48 h) kynurenine administration led to significant changes in protein expression in the spleen, including multiple changes involved with cytoskeletal rearrangements associated with cell motility. Kynurenine administration resulted in several expression level changes in proteins associated with heat shock protein 90 (HSP90), a chaperone for the aryl-hydrocarbon receptor (AHR), which is the primary kynurenine metabolite receptor. We propose that cells with high levels of Quin are those that are currently releasing kynurenine pathway metabolites as well as accumulating Quin for sustained NAD+ synthesis from tryptophan. Further, we propose that the kynurenine pathway may be linked to the regulation of cell motility in immune and cancer 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.

Pseudomonas aeruginosa resistance of monosaccharide-functionalized glass surfaces.

Preventing microorganism colonization on a surface is a great challenge in the conception of medical, food and marine devices. Here, we describe the formation of carbohydrate functionalized glass surfaces with D-glucose, D-galactose and D-mannose and how they efficiently affected the bacterial attachment. The carbohydrate entities were covalently attached to the pre-functionalized surface by click chemistry thanks the copper catalysed alkyl-azide cycloaddition. Water contact angle and X-ray photoelectron spectroscopy characterisations showed a homogeneous and quantitative cycloaddition at the scale of microorganisms. The adhesion assays with Pseudomonas aeruginosa, used as model of opportunistic pathogen, indicated a significant diminution of almost 40% of the bacterial accumulation on glycosidic surfaces with respect to initial surface. This activity was further compared with a surface presenting a simple hydroxyl residue. Exploration of specific interactions through Lectin A deficient Pseudomonas aeruginosa mutant strain provided new evidences that Lectin A was involved in biofilm maturation, rather than bacterial attachment. Subsequently, the determination of surface free energy and the adhesion free energy between surfaces and bacterial cell wall showed that the adhesion was thermodynamically unfavourable.

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.

Sex differences in stress and immune responses during confinement in Antarctica.

BACKGROUND: Antarctica challenges human explorers by its extreme environment. The effects of these unique conditions on the human physiology need to be understood to best mitigate health problems in Antarctic expedition crews. Moreover, Antarctica is an adequate Earth-bound analogue for long-term space missions. To date, its effects on human physiology have been studied mainly in male cohorts though more female expeditioners and applicants in astronaut training programs are selected. Therefore, the identification of sex differences in stress and immune reactions are becoming an even more essential aim to provide a more individualized risk management. METHODS: Ten female and 16 male subjects participated in three 1-year expeditions to the German Antarctic Research Station Neumayer III. Blood, saliva, and urine samples were taken 1-2 months prior to departure, subsequently every month during their expedition, and 3-4 months after return from Antarctica. Analyses included cortisol, catecholamine and endocannabinoid measurements; psychological evaluation; differential blood count; and recall antigen- and mitogen-stimulated cytokine profiles. RESULTS: Cortisol showed significantly higher concentrations in females than males during winter whereas no enhanced psychological stress was detected in both sexes. Catecholamine excretion was higher in males than females but never showed significant increases compared to baseline. Endocannabinoids and N-acylethanolamides increased significantly in both sexes and stayed consistently elevated during the confinement. Cytokine profiles after in vitro stimulation revealed no sex differences but resulted in significant time-dependent changes. Hemoglobin and hematocrit were significantly higher in males than females, and hemoglobin increased significantly in both sexes compared to baseline. Platelet counts were significantly higher in females than males. Leukocytes and granulocyte concentrations increased during confinement with a dip for both sexes in winter whereas lymphocytes were significantly elevated in both sexes during the confinement. CONCLUSIONS: The extreme environment of Antarctica seems to trigger some distinct stress and immune responses but-with the exception of cortisol and blood cell counts-without any major relevant sex-specific differences. Stated sex differences were shown to be independent of enhanced psychological stress and seem to be related to the environmental conditions. However, sources and consequences of these sex differences have to be further elucidated.

Lectin-Functionalized Composite Hydrogels for "Capture-and-Killing" of Carbapenem-Resistant Pseudomonas aeruginosa.

Infections with multiresistant pathogens are a leading cause for mortality worldwide. Just recently, the World Health Organization (WHO) increased the threat rating for multiresistant Pseudomonas aeruginosa to the highest possible level. With this background, it is crucial to develop novel materials and procedures in the fight against multiresistant pathogens. In this study, we present a novel antimicrobial material, which could find applications as a wound dressing or antimicrobial coating. Lectins are multivalent sugar-binding proteins, which can be found in a variety of plants and bacteria, where they are associated with biofilm formation. By immobilizing lectin B on a protein-based hydrogel surface, we provided the hydrogel with the ability to immobilize ("catch") pathogens upon contact. Furthermore, another hydrogel layer was added which inhibits biofilm formation and releases a highly potent antimicrobial peptide to eradicate microorganisms ("kill"). The composite hydrogel showed a high antimicrobial activity against the reference strain Pseudomonas aeruginosa PAO1 as well as against a carbapenem-resistant clinical isolate (multiresistant Gram-negative class 4) and may thus represent a novel material to develop a new type of antimicrobial wound dressings to prevent infections with this problematic pathogen of burn or other large wounds.

Alteration of salivary glycopatterns in oral lichen planus.

BACKGROUND: Oral lichen planus (OLP) is one of the most common oral mucosal lesions affecting 0.5-2% of the adult population. It is difficult to distinguish between OLP and other oral mucosal diseases. Structural changes in the glycans of saliva proteins might be reliable indicators of OLP. However, little is known about the alteration of salivary glycopatterns during OLP. OBJECTIVE: We aimed to investigate the alterations of salivary protein glycosylation related to OLP. MATERIAL AND METHODS: Twenty-eight patients with OLP and 30 age- and sex-matched healthy volunteers (HVs) were enrolled in the test group to probe the difference of salivary glycopatterns using lectin microarrays. The lectin blotting were further utilized to validate the expression of certain glycans. RESULTS: The glycoproteins recognized by three lectins [Aleuria aurantia lectin (AAL); Phytolacca americana (PWM); Phaseolus vulgaris agglutinin (E + L), (PHA-E + L)] were mainly increasing in the saliva of OLP. Meanwhile, these glycoproteins also exhibited significant age-associated alterations. CONCLUSIONS: This study provided a new basic insight into salivary glycopatterns in OLP and helped to develop new potential biomarkers for diagnosis of OLP.

Lectin-mediated reversible immobilization of human cells into a glycosylated macroporous protein hydrogel as a cell culture matrix.

3D cell culture is a helpful approach to study cell-cell interaction in a native-like environment, but is often limited due the challenge of retrieving cells from the material. In this study, we present the use of recombinant lectin B, a sugar-binding protein with four binding cavities, to enable reversible cell integration into a macroporous protein hydrogel matrix. By functionalizing hydrogel precursors with saccharose, lectin B can both bind to sugar moieties on the cellular surface as well as to the modified hydrogel network. Confocal microscopy and flow cytometry analysis revealed cells to be integrated into the network and to adhere and proliferate. Furthermore, the specificity and reversibility was investigated by using a recombinantly produced yellow fluorescent - lectin B fusion protein and a variety of sugars with diverging affinities for lectin B at different concentrations and elution times. Cells could be eluted within minutes by addition of L-fucose to the cell-loaded hydrogels to make cells available for further analysis.

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.