Immune Cell Reactivity Research Articles

Omental Milky Spots Develop in the Absence of Lymphoid Tissue-Inducer Cells and Support B and T Cell Responses to Peritoneal Antigens

The omentum is a site of B1 cell lymphopoiesis and immune responsiveness to T cell-independent antigens. However, it is unknown whether it supports immune responses independently of conventional lymphoid organs. We showed that the omentum collected antigens and cells from the peritoneal cavity and supported T cell-dependent B cell responses, including isotype switching, somatic hypermutation, and limited affinity maturation, despite the lack of identifiable follicular dendritic cells. The omentum also supported CD4+ and CD8+ T cell responses to peritoneal antigens and recruited effector T cells primed in other locations. Unlike conventional lymphoid organs, milky spots in the omentum developed in the absence of lymphoid tissue-inducer cells, but required the chemokine CXCL13. Although the lymphoid architecture of milky spots was disrupted in lymphotoxin-deficient mice, normal architecture was restored by reconstitution with lymphotoxin-sufficient hematopoietic cells. These results indicate that the milky spots of the omentum function as unique secondary lymphoid organs that promote immunity to peritoneal antigens.

Isoform-specific regulation of immune cell reactivity by the catalytic subunit of protein kinase A (PKA)

There are two major genes encoding the catalytic subunits of protein kinase A, Cα and Cβ. The functional significance of these isoforms is enigmatic. Lymphoid cells of the immune system express both Cα and Cβ. In this study we tested the role of Cα and Cβ in regulating immune cell reactivity to antigens using mice carrying a targeted disruption of the Cα and Cβ gene respectively. Cα and Cβ ablation both resulted in a 50% reduction in PKA-specific kinase activity and the level of PKA type I but not PKA type II. Moreover, despite that C subunit ablation did not affect immune cell development and homeostasis, Cα but not Cβ ablation augmented expression of the activation marker CD69 on lymphocytes. CD69 induction coincided with immune cell hyperresponsiveness and was associated with reduced sensitivity to cAMP-mediated inhibition of anti-CD3 induced T cell proliferation. Our results imply that Cα is required for normal immune cell reactivity and demonstrates isoform-specific effects and non-redundant functions of C subunit isoforms expressed in the same cell.

Inhibition of CD4+ T lymphocyte binding to fibronectin and immune-cell accumulation in inflammatory sites by non-peptidic mimetics of Arg-Gly-Asp.

The Arg-Gly-Asp (RGD) cell adhesion motif has been demonstrated in various studies to play a pivotal role in leucocyte and platelet interactions with plasma and extracellular matrix (ECM) glycoproteins. The recognition of the RGD sequence is mediated by heterodimeric receptors designated integrins of the beta 1 subfamily, expressed on distinct cell types, including T lymphocytes. We have recently shown that flexible non-peptidic mimetics of RGD, in which the two ionic side groups were separated by a linear spacer of 11 atoms, bound specifically to the platelet integrin alpha 11b beta 3, and inhibited T cell-mediated immune responses. The present study was designed to (i) further characterize the structural requirements for RGD interactions with CD4+ T cells, and (ii) examine the mechanisms by which the RGD mimetics interfere with immune cell reactivity in vivo. We now report that freezing the conformational degrees of freedom in the spacer chain, which fixes the relative orientation of the guanidinium and carboxylate side groups in a favourable manner, results in a higher level of inhibition of T cell binding to immobilized fibronectin, an RGD-containing ECM glycoprotein. In vivo, treatment of mice with relatively low doses of the RGD mimetics, but not the RGD peptide, inhibited the elicitation of an adoptively transferred DTH reaction. This inhibition was achieved by direct impairment of the ability of antigen-primed lymph node cells to migrate and accumulate in inflammatory sites. Hence, we suggest that the design and production of non-peptidic mimetics of RGD offers a novel approach to study defined parameters related to the structure-function requirements of small adhesion epitopes. Furthermore, this approach could be used therapeutically to inhibit pathological processes which depend on RGD recognition.

Expression of 25 hydroxyvitamin D3-1α-hydroxylase in human endometrial tissue

1,25(OH)2D3 (calcitriol) has been shown to play an important role in cell proliferation, differentiation and immune responsiveness. The enzyme responsible for calcitriol synthesis 25 hydroxyvitamin D3-1α-hydroxylase (1α-OHase) has been reported in many human tissues. The aim of this study was to investigate the expression of 1α-OHase in gynaecological tissues. Using a highly specific nested touchdown PCR we examined the expression of 1α-OHase in normal and malignant endometrial tissue and in human endometrial Ishikawa cells. In addition, we analyzed the protein expression of 1α-OHase by Western blot. The expression of 1α-OHase in normal and malignant endometrial tissue and Ishikawa cells was detected and splice variants of the enzyme in Ishikawa cells were identified. These data suggest an alternative splicing of 1α-OHase in malignant endometrial tissue and cells. We postulate that the expression of 1α-OHase gene variants may contribute to the antiproliferative effects of calcitriol. In conclusion, the modulation of the 1α-OHase opens up a new target for vitamin D3 related therapies in endometrial cancer.

Modulatory effects of several herbal extracts on avian peripheral blood cell immune responses

Standardized ethanol extracts of Allium sativum (garlic), Glycyrrhiza glabra (licorice), Plantago major (plantain) and Hippophae rhamnoides (sea buckthorn) were assessed for their effects on cellular immunity in laying hens. Birds (n = 25) had blood samples taken and both specific and non-specific immune cell responsiveness were evaluated by a leukocyte proliferation assay, carbon clearance test and SRBC phagocytosis in monocyte-derived macrophage cultures. Licorice and sea buckthorn (50 microg/mL) clearly enhanced the macrophage membrane function (p < 0.05 and p < 0.01, respectively). Dual effects on circulating phagocytes were revealed for plantain and sea buckthorn, while garlic at 200 microg/mL impaired the phagocytic capacity of blood cells. None of the tested extracts showed mitogenic properties, but high concentrations of sea buckthorn (400 microg/mL) inhibited leukocyte proliferation. Small concentrations (20 microg/mL) of licorice proved the co-mitogenic potential for both T and B avian lymphocytes (p < 0.05). Certain extracts definitely enhanced the fowl innate and/or specific cell immunity and may therefore improve host resistance in poultry. Considering the chicken as an important non-mammalian model that also serves as an available laboratory approach for some human diseases, herbs exerting immunomodulatory properties may find relevant clinical applications.

T lymphocytes and dendritic cells are activated by the deletion of peroxiredoxin II (Prx II) gene

Peroxiredoxin II (Prx II) is a member of antioxidant enzyme family and it plays a protective role against oxidative damage. Constitutive production of endogenous reactive oxygen species was detected in spleen and bone marrow cells lacking Prx II. Here, we investigated the role of Prx II in immune responses. The total number of splenocytes (especially, the population of S-phase cells and CD3 + T cells) was significantly higher in Prx II −/− mice than in wild type. Number of peripheral blood mononuclear cells (PBMCs) in Prx II −/− mice was also higher than wild type. Differentiation of Prx II −/− mouse bone marrow cells into CD11c-positive dendritic cells was greater than that of wild type. Transplantation of Prx II −/− bone marrow cells into wild type mice increased PBMCs in blood and bone marrow-derived dendritic cells. Prx II deletion enhances concanavalin A (ConA)-induced splenocyte proliferation and mixed lymphocyte reaction (MLR) activity of bone marrow-derived CD11c-positive dendritic cells to stimulate recipient splenocytes. Collectively, these data suggest that Prx II inhibits the immune cell responsiveness, which may be regulated by scavenging the low amount of reactive oxygen species (ROS).

Linking the “two-hit” response following injury to enhanced TLR4 reactivity

Severe injury can initiate an exaggerated systemic inflammatory response and multiple organ failure (MOF) if a subsequent immune stimulus, "second hit", occurs. Using a mouse thermal injury model, we tested whether changes in innate immune cell reactivity following injury can contribute to the development of heightened inflammation and MOF. Using high-purity Escherichia coli lipopolysaccharide (LPS) to selectively stimulate Toll-like receptor 4 (TLR4), we demonstrate augmented interleukin (IL)-1beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 production by 1 day but particularly, at 7 days after injury. The in vivo significance of enhanced TLR4 responsiveness was explored by challenging sham or burn mice with LPS at 1 or 7 days after injury and determining mortality along with in vivo cytokine and chemokine levels. Mortality was high (75%) in LPS-challenged burn but not sham mice at 7 days, although not at 1 day, after injury. Death was associated with leukocyte sequestration in the lungs and livers along with increased proinflammatory cytokine and chemokine levels in these organs. Blocking TNF-alpha activity prevented this mortality, suggesting that excessive TNF-alpha production contributes to this lethal response. These findings demonstrate the potential lethality of excessive TLR4 reactivity after injury and provide an explanation for the exaggerated inflammatory response to a second hit, which can occur following severe injury.

Clinical Implications of T Cell Receptor Repertoire Analysis after Allogeneic Stem Cell Transplantation

Stem cell transplantation (SCT) constitutes a major challenge to the immune system. Long-term impairment of immunity against various common infectious stimuli leads to increased susceptibility to infectious diseases; in contrast, an immune response against the recipient may cause the devastating graft-versus-host disease (GvHD). Recovery of the immune system (both qualitative and quantitative) after SCT is perhaps the most important factor in determining the clinical outcome. Consequently, immune reconstitution has been extensively studied using different approaches, including quantitative analysis of immune cells as well as their phenotypic characterization. Analysis of diversity and clonality is an important tool in determining competence of the immune system, assuming that a broad diversity assures efficient response to different stimuli and clonal dominance reflects ongoing, potentially relevant immune responses. Detailed analysis of the immune repertoire through the flow cytometric and molecular study of the T cell receptor repertoire has been applied to gain quantitative and qualitative insights about the T cell immune competence and responsiveness. After SCT, a contraction of the T cell pool and a reduction in T cell receptor diversity is clearly associated with clinical immunodeficiency. Reconstitution of the immune system is often characterized by dominance of oligoclonal T cell populations, reflecting specific antigen-driven immune responses. Detailed characterization of T lymphocytes by T cell receptor analysis is possible, and may lead to the identification of individual clones involved in specific immune reactions, such as alloresponses in GvHD, the closely related graft-versus-leukemia effect and opportunistic viral agents such as CMV or EBV.

Effect of tetrachloromethane on the immune system.

Acute poisoning with tetrachloromethane in a dose of 0.75 LD(50) suppressed humoral and cell immune reactions in Wistar rats. Immunotoxicity of tetrachloromethane is realized via initiation of lipid peroxidation and inhibition of acetylcholine esterase in T lymphocytes and alpha-naphthyl AS-acetate esterase and alpha-naphthyl butyrate esterase in splenocytes.

The role of nonsteroidal antiinflammatory drugs in cancer chemoprevention

Epidemiological and laboratory studies, as well as human clinical intervention trials, strongly support a protective role for nonsteroidal antiinflammatory drugs (NSAIDs) in preventing the development of cancer. For example, NSAIDS are effective for reducing existing polyps in patients with familial adenomatous polyposis and reduce the tumor burden in several animal models of colorectal cancer. Many studies suggest that cyclooxygenase, especially COX-2, can be causally connected to carcinogenesis via modulation of the synthesis of prostaglandins and other eicosanoids, and subsequent effects on cell proliferation, angiogenesis, tumor growth, immune responsiveness and inflammatory reactions. it is therefore reasonable to assume that NSAIDS as inhibitors of COX-2 would be good candidates for cancer prevention, or even treatment. Animal studies and in vitro cell culture bioassays further demonstrated that cyclooxygenase[ independent actions of NSAIDs could also contribute to their anticancer activities. These studies showed that NSAIDS can affect the expression and functions of many transcription factors related to oncogenesis, including hypoxia-inducible factor-1 (HIF-1), GATA-6, NF-κB, β-catenin/TCF-4, early growth response-1 (Egr-1) and the androgen receptor.

Early glial cell reactivity in experimental retinal detachment: effect of suramin.

In a rabbit model of retinal detachment, early Müller glial cell reactivity was monitored-specifically, changes in membrane features-to determine whether these changes involve an upregulation of purinergic P2 receptor-mediated responses and whether all or some of these alterations could be blocked by suramin or pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS). In addition, the immune cell reactivity (microglial cells and blood-derived immune cells) was monitored. A local retinal detachment was induced by subretinal injection of a sodium hyaluronate solution. Three, 24, 48, and 72 hours after surgery, Müller cells were acutely isolated, and patch-clamp records of the whole-cell potassium currents were made. The presence of P2 receptor-mediated responses was determined by measuring extracellular adenosine triphosphate (ATP)-induced membrane current increases, and by recording of ATP-induced calcium responses at the vitreal surface of retinal wholemounts. The density of isolectin B(4)-labeled immune cells was determined in the nerve fiber layer of retinal wholemounts. Within 24 hours of detachment, Müller cell reactivity was evident. The cells downregulated the density of their inwardly rectifying potassium currents to 60% and 47% of the control value at 48 hours and 72 hours of detachment, respectively. This downregulation was accompanied by an enhanced incidence of cells which showed calcium and current responses after ATP application (control: 14%; 24 hours of detachment: 42%; 72 hours of detachment: 80%). Müller cell hypertrophy was apparent at 48 and 72 hours of detachment. Application of suramin during surgery inhibited the downregulation of potassium currents, but not the elevated responsiveness to extracellular ATP; PPADS had no effect. Suramin also inhibited the inflammatory response that was induced by the surgical procedure and that was apparent by the increased number of immune cells. Reactive responses of Müller cells occur within 24 hours of detachment. Suramin inhibits several (but not all) reactive glial alterations and therefore may represent one candidate for further investigations in the search for drugs that limit detrimental effects of immune cell activation and Müller cell gliosis during retinal detachment.

Proteins of the Rpf family: immune cell reactivity and vaccination efficacy against tuberculosis in mice.

It was shown recently that Mycobacterium tuberculosis expresses five proteins that are homologous to Rpf (resuscitation promoting factor), which is secreted by growing cells of Micrococcus luteus. Rpf is required to resuscitate the growth of dormant Micrococcus luteus organisms, and its homologues may be involved in mycobacterial reactivation. Mycobacterial Rpf-like products are secreted proteins, which makes them candidates for recognition by the host immune system and anti-Rpf immune responses potentially protective against reactivated tuberculosis. Here we report that the Rpf protein itself and four out of five of its mycobacterial homologues, which were administered as subunit vaccines to C57BL/6 mice, are highly immunogenic. Rpf-like proteins elicit immunoglobulin G1 (IgG1) and IgG2a responses and T-cell proliferation and stimulate production of gamma interferon, interleukin-10 (IL-10), and IL-12 but not IL-4 or IL-5. Both humoral and T-cell responses against these antigens show a high degree of cross-reactivity. Vaccination of mice with Rpf-like proteins results in a significant level of protection against a subsequent high-dose challenge with virulent M. tuberculosis H37Rv, both in terms of survival times and mycobacterial multiplication in lungs and spleens.

CpG immuno-stimulatory motifs enhance humoral immune responses against hepatitis C virus core protein after DNA-based immunization.

Chronic HCV infection is associated with a high morbidity and mortality rate, and currently a prophylactic or therapeutic vaccine is not available. DNA-based immunization is a powerful method to generate cellular and humoral immune responses. However, DNA immunization against HCV core results only in a weak humoral immune response demonstrated in several studies. Therefore, co-immunization with a novel adjuvant may enhance such potentially important immune responses. We examined whether unmethylated CpG motifs in the form of oligodeoxynucleotides (ODN) or E. coli DNA can act as adjuvants for a DNA vaccination approach, since CpG motifs have been shown to stimulate the innate immune system as well as B and T cell immune reactivity. The present study demonstrates that CpG motifs enhance in vivo antibody levels after DNA immunization against HCV core. However, despite some in vitro activity of CpG motifs, no enhancement of T cell responses in vivo was observed after immunization with HCV plasmid DNA and CpG motifs in mice. Our results suggest that co-immunization with CpG-ODN may strengthen humoral immune responses but show no potential effect as an adjuvant to induce cellular immunity against HCV core.

Treatment of adjuvant arthritis with granulocyte-colony stimulating factor and peptide derived from heat shock protein 65

Adjuvant arthritis in Lewis rats is induced by the subcutaneous injection of Mycobacterium tuberculosis in mineral oil, and the predominant T cell immune reactivity is against the heat shock protein 65 derived peptide 176–190. We treated Lewis rats with human recombinant G-CSF followed by (i.v) administration of peptide 176–190 after induction of adjuvant arthritis (AA), and observed decreased disease severity, joint destruction, new bone formation and joint ankylosis. Treatment with G-CSF alone was also effective, but to a lesser extent. In addition, we found that splenocytes from rats treated with G-CSF had reduced antigen presenting capacity compared with splenocytes from vehicle treated rats. Primed lymph node cells from G-CSF plus peptide treated rats showed a marked reduction in proliferation and secretion of IFN-γ after stimulation with the heat shock protein peptide in vitro as compared to controls.

Vitamin D as immunomodulatory therapy for kidney transplantation1

Vitamin D (1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)]) has been studied in the past for its immunosuppressive properties, and, in that context, it may also have potential utility as an immunomodulatory agent for transplantation. A number of studies have demonstrated that 1alpha,25-(OH)(2)D(3) or its analogs regulate immune cell proliferation, differentiation, and responsiveness. A burgeoning number of studies have also explored using 1alpha,25-(OH)(2)D(3) and its analogs directly as therapy in animal models of kidney transplantation with success in prolonging allograft function and preventing acute rejection. Some of these in vivo effects may well be caused by alterations in immune cell function, but it is also possible that exogenous 1alpha,25-(OH)(2)D(3) and its analogs are altering the intragraft milieu as well, specifically through changes in the TGF-beta signaling cascade. Such provocative data and the availability of newer 1alpha,25-(OH)(2)D(3) analogs that may limit side effects (e.g. hypercalcemia) have created interest in examining this secosteroid clinically in kidney transplantation.

Tumor necrosis factor-α production in whole blood after cardiopulmonary bypass: Downregulation caused by circulating cytokine-inhibitory activities

Objectives: Cardiopulmonary bypass is associated with the release of proinflammatory cytokines (tumor necrosis factor α, interleukin 1β, interleukin 6, and interleukin 8) and anti-inflammatory cytokines (interleukin 10 and transforming growth factor β1). On the one hand this cytokine release is related to the postoperative systemic inflammatory response syndrome, and on the other hand it is related to deterioration of the immune system, for example in monocyte or polymorphonuclear neutrophil function, leading to an increased susceptibility to infections. To gain further insight into the alterations of immune cell reactivity and possible regulatory mechanisms, we studied lipopolysaccharide-induced tumor necrosis factor α synthesis in whole blood from cardiac surgical patients. Methods: Fifteen patients undergoing elective heart surgery with cardiopulmonary bypass were included in the study. Ex vivo lipopolysaccharide-induced tumor necrosis factor α synthesis was measured in a whole blood assay before, during, and after bypass. Corresponding tumor necrosis factor α messenger RNA levels were determined by semiquantitative reverse transcriptase-polymerase chain reaction. In addition, the influence of patient serum on whole blood responsiveness and its relationship to anti-inflammatory cytokines were evaluated in vitro. Results: Tumor necrosis factor α synthesis was significantly reduced after 30 minutes of cardiopulmonary bypass and showed the lowest values at the end of bypass (mean ± SD 0.109 ± 0.105 ng/106 white blood cells after 30 minutes of bypass and 0.050 ± 0.065 ng/106 white blood cells at the end of bypass, vs 0.450 ± 0.159 ng/106 white blood cells preoperatively, P <.001). As a further indication of reduced cytokine biosynthesis, diminished messenger RNA levels for tumor necrosis factor α were detected. Serum withdrawn from patients at the end of cardiopulmonary bypass reduced tumor necrosis factor α synthesis in heterologous blood from healthy volunteers highly significantly to 39.93% ± 23.18% relative to control serum (P =.005) and preoperatively drawn serum (P =.024). This effect was dose dependent and was not specific for lipopolysaccharide-induced tumor necrosis factor α synthesis. Anesthesia and heparin administration did not influence tumor necrosis factor α production significantly. Ex vivo tumor necrosis factor α synthesis was negatively related to interleukin 10 serum levels, positively but weakly related to interleukin 4, and was not related to transforming growth factor β1 (Spearman correlation coefficients −0.565, P <.001, 0.362, P <.001, and −0.062, P =.460, respectively). However, interleukin 10 levels in patient serum after cardiopulmonary bypass were 300-fold below the quantities needed for half-maximal inhibition of tumor necrosis factor α synthesis in vitro. Moreover, the inhibitory activity could not be removed by immune absorption of interleukin 10. Conclusions: These results suggest that during cardiac operations cytokine-inhibitory serum activities are released or newly formed. These activities could not be explained by the actions of interleukins 4 and 10 or transforming growth factor β1. Although their exact nature remains undetermined, these substances may contribute to the diminished immune cell functions after cardiopulmonary bypass and thus need further characterization.J Thorac Cardiovasc Surg 2002;124:608-17

Cosmetics for the elderly

Aging remains an enigma and the visible modifications that slowly appear in aging skin are provided by a gradual reduction in the size and number of cells and a decrease in the rate of many organic functions. Skin aging is the result of a programmed multistep, time-dependent continuous phenomenon at the cellular and molecular level.1,2 Little attention has been given to the issue of appearance in the elderly until recently. Skin functions that decline with age are cell replacement, injury response, barrier function, chemical clearance, sensory perception, immune and vascular responsiveness, thermoregulation, sweat production, sebum production, and vitamin D production.3 Successful aging is characterized by good mental health, adaptative psychosocial functioning, and life satisfaction.4 In the United States in 1998, the older population (65 years or older) represented 12.7% of the total: approximately one in every eight Americans. Since 1900, the percentage of Americans older than 65 years of age has more than tripled (4.1% in 1900 to 12.7% in 1998), and the number has increased eleven times (from 3.1 million to 34.4 million). By 2030, the estimate is that there will be approximately 70 million senior citizens, more than twice the number in 1998. People over 65 years of age are expected to represent 13% of the population in the year 2000 but will be 20% by 2030. The 40 million “baby boomers” born in the United States between 1946 and 1963 are approaching that stage of life in which, unfortunately, youthful good looks are becoming more of a memory than a reality. They obviously cannot remain forever young, but cosmetic innovations through scientific research are progressing very rapidly. Patients are able to significantly delay the visible aging process by practicing preventive skin care and taking advantage of the many chemical treatments available for facial rejuvenation.5 Cutaneous aging provides a visible model of the interaction between endogenous and exogenous (UV light) factors. Recently, much interest has been directed toward defining the aging processes in the skin, aiming to highlight areas such as environmental changes and their influence in skin aging and carcinogenesis.6,7 Clinically detectable, age-associated cutaneous changes result from two independent processes: chronologic aging and actinic irradiation (Fig 1). Several lines of evidence suggest that these two processes have different biologic, biochemical, and molecular mechanisms.8 Understanding these events at all these levels has unfortunately not been paralleled by clinical and therapeutic advances about common, troublesome skin problems of old age, such as cancer, xerosis, and pruritus. These conditions remain major causes of morbidity and are yet poorly explained.6,7 The main skin functions include protection, excretion, secretion, absorption, thermoregulation, pigmentogenesis, accumulation, sensory perception, and regulation of immunological processes; all of these functions are altered with the passing of time. Physiological changes include impairment of the barrier function, decreased turnover of epidermal cells, reduced numbers of keratinocytes and fibroblasts, and a reduced vascular network particularly around hair bulbs and glands, leading to fibrosis and atrophy, decrease of hair and nail growth, retardation in vitamin D synthesis, decline of Langerhans’ cells density, and impairment of the immune response.9–12 There is also a decrease in the function of Meissner’s and Pacinian corpuscles. Increased production of free radicals is observed and cutaneous permeability to chemical substances also changes, while water-binding capacity of the stratum corneum is reduced.8,13–15 Epidermal changes associated with aging involve the flattening of its underside, a reduction in the number of Langerhans’ cells and of melanocytes, and a decline in the number of synthesized melanosomes, leading to reduced pigmentation. Dermal changes are represented by a reduction in collagen and elastic fibers, fibroblasts, mast cells and macrophages, and dilation of the lymphatic channels. The most affected cells in the senescence process are keratinocytes and fibroblasts. The dermal–epidermal junction is flattened and the number of hair follicles decrease with age, although their structure remains unchanged.10,11,16–20 Colorimetric measurements show that elderly skin is darker than young skin even in areas not exposed to the sun. Epidermal atrophy and increased transparency From the Sector of Dermatology, HUCFF-UFRJ and School of Medicine, Universidade Federal do Rio de Janeiro, (MRS and SCSC) and the HUPEUERJ and Sector of Dermatology, School of Medicine, Universidade do Estado do Rio de Janeiro, (SCSC) Rio de Janeiro, Brazil. Address correspondence to Marcia Ramos-e-Silva, MD, Rua Sorocaba 464/205, 22271-110-Rio de Janeiro, Brazil. E-mail address: ramos.e.silva@dermato.med.br.

Improving the in vitro antigen specific T cell proliferation assay: the use of interferon-alpha to elicit antigen specific stimulation and decrease bystander proliferation

The measurement of the proliferative response of primed T cells to an antigenic stimulus (lymphocyte transformation assay: LTT) is commonly used for determining T cell immune responsiveness. However, the ratio between the spontaneous and the antigen-triggered response (stimulation index) is frequently quite low (<3–5) making the interpretation difficult. We modified the assay by the addition of interferon-α and the use of fresh autologous serum instead of human AB pool serum. These measures significantly enhanced the stimulation index following stimulation with tetanus toxoid, Candida albicans and tick-borne encephalitis (TBE) viral antigen in studies of sensitized patients. There was no concomitant increase in false positive results. Kinetic studies showed a reduced nonspecific background proliferation of non-stimulated cultures particularly between days 4 and 6 of culture. Furthermore, the positive effect of interferon-α were confirmed in studies of patients with contact allergy to nickel and gold. We conclude that this modified form of proliferation assay significantly increases the signal to noise ratio which can be attained. This may be of particular value when looking at T cell responses in immunocompromised patients or in diagnostic attempts to detect very low frequencies of antigen-specific T cells.

Immune responses to P.falciparum-MSP1 antigen: lack of correlation between antibody responses and the capacity of peripheral cellular immune effectors to respond to this antigen in vitro

Protective immunity to P. falciparum blood stage infection is thought to be dependent on IgG antibodies, although the mechanisms that underlie such immunity are not clearly understood. One of the antigens thought to be involved in this protective response is MSP1. The present study has examined the levels and distribution of IgG (and IgM) antibodies to the C-terminal 19 kDa fragment of MSP1 in plasma from P. falciparum immune adult Senegalese and the capacity of the peripheral blood mononuclear cells from these patients to either proliferate or secrete IFN-γ, IL-10 or IL-4 in vitro in response to this antigen. Specific antibodies were found in 74% of individuals’ plasma; 44% of mononuclear cells proved capable of proliferating in vitro and IFN-γ, IL-10 and IL-4 were detected in 37, 23 and 0% of culture supernatants, respectively. No significant association was found between the presence of antibodies and immune cell reactivity under the culture conditions used. This study emphasizes the complexity of the mechanisms responsible for the sustained production of potentially protective antibodies in response to proposed T-cell dependent P. falciparum blood stage antigens.

Immunosuppressive Phenotype of Corticotropin-Releasing Factor Transgenic Mice Is Reversed by Adrenalectomy

Stress elicits a wide range of physiological changes involving the nervous, endocrine, and immune systems. Corticotropin-releasing factor (CRF) plays a key role in orchestrating this response, activating both the sympathetic nervous system and the hypothalamic–pituitary–adrenal axis, resulting in release of corticosteroids. The present study examines the immunological phenotype and responsiveness of CRF-transgenic (CRF-Tg) mice. The immune system of the CRF-Tg animals has profound changes compared to littermate controls, including a marked reduction in both cell number and immune responsiveness. There were also phenotypic changes in the lymphocytic composition of the various lymphoid organs, most notably in the spleen, where CRF-Tg mice had a greater percentage of T lymphocytes compared to littermate controls. Adrenalectomy of CRF-Tg reversed the immunological phenotype observed and restored immune responsiveness. These results demonstrate that CRF overexpression leads to profound impairment on lymphocyte development and function mediated via corticosteroids.