Multicenter Evaluation of the New Automated Enzymun-Test Anti-HIV 1 + 2 + Subtyp O

An international multicenter study was performed to evaluate a new, automated human immunodeficiency virus (HIV) third-generation antibody assay. The Enzymun-Test Anti-HIV 1 + 2 + Subtyp O showed 100% sensitivity and 99.8% specificity among 11,172 samples from hospitalized patients and blood donors. For early HIV antibody detection in seroconversion panels, Enzymun-Test showed a sensitivity equivalent to that of the Abbott Recomb. HIV-1/HIV-2 3rd Gen. assay.     

In Vitro Expansion of T-Cell-Receptor Vα2.3+ CD4+ T Lymphocytes in HLA-DR17(3), DQ2+ Individuals upon Stimulation with Mycobacterium tuberculosis

The T-cell receptor (TCR) Valpha/beta gene product expression upon in vitro stimulation with mycobacteria was investigated to assess whether T-cell proliferation was associated with any specific TCR V gene usage. T-cell-enriched populations from peripheral blood of Mycobacterium bovis BCG-vaccinated healthy blood donors were stimulated in vitro with live or killed M. tuberculosis or with a soluble extract thereof. TCR Valpha/beta repertoire analysis of reactive CD4(+) and CD8(+) T cells revealed a selective HLA-DR17(3), DQ2-restricted expansion of Valpha2.3(+) CD4(+) T cells upon stimulation with live M. tuberculosis or its soluble extract. Third-complementarity-determining-region (CDR3) length analysis of the expanded Valpha2.3(+) T cells indicated an oligoclonal pattern with short CDR3 lengths in six of seven HLA-DR17(3), DQ2(+) individuals tested. In addition, Valpha/Vbeta repertoire analysis of T lymphocytes from a DR17(3), DQ2(+) donor before and after BCG vaccination revealed that positivity of skin test reactivity was associated with expansion of Valpha2.3(+) CD4(+) T lymphocytes with preferential use of a short CDR3 peak length after in vitro stimulation. Separation of M. tuberculosis soluble extract by fast protein liquid chromatography (FPLC) purification indicated that fractions corresponding to molecular masses of 60 to 70 and 15 to 25 kDa were particularly effective in eliciting Valpha2.3(+) CD4(+) T-cell expansion.     

CD4+ CD25+ T-Cell Production in Healthy Humans and in Patients with Thymic Hypoplasia

Regulatory T cells are found primarily in the CD4⁺ CD25⁺ fraction of T cells and play an important role in the prevention of autoimmunity. We examined CD4⁺ CD25⁺ T cells in 33 healthy children and adults and compared them to a population with an inherited form of thymic hypoplasia and a predisposition to autoimmune disease. Absolute numbers of CD4⁺ CD25⁺ T cells were markedly higher in healthy infants than in infants with chromosome 22q11.2 deletion syndrome.     

Expanded CD14+ CD16+ Monocyte Subpopulation in Patients with Acute and Chronic Infections Undergoing Hemodialysis

Infections are frequent complications in end-stage renal failure patients undergoing hemodialysis (HD), and peripheral blood monocytes are important cells in host defense against infections. The majority of circulating monocytes express high levels of lipopolysaccharide receptor antigen CD14 and are negative for the immunoglobulin Fcγ receptor type III (CD16). We studied the occurrence of a minor subpopulation coexpressing low levels of CD14 together with CD16 in HD patients. In healthy controls CD14⁺ CD16⁺ monocytes account for 8% ± 4% of CD14⁺ monocytes, with an absolute number of 29 ± 14 cells/μl. In stable HD patients the CD14⁺ CD16⁺ subpopulation was significantly elevated (14% ± 3%, or 66 ± 28 cells/μl), while the number of CD14⁺⁺ monocytes (monocytes strongly positive for CD14) remained constant. In HD patients suffering from chronic infections a further rise in CD14⁺ CD16⁺ monocytes was observed (128 ± 71 cells/μl; P < 0.01) such that this subpopulation constituted 24% of all blood monocytes. In contrast, numbers of CD14⁺⁺ cells did not change compared to those for stable HD patients, indicating that the CD14⁺ CD16⁺ monocyte subpopulation was selectively expanded. During acute infections the CD14⁺ CD16⁺ cell subpopulation always expanded. A whole-blood assay revealed that CD14⁺ CD16⁺ monocytes exhibited a higher phagocytosis rate for Escherichia coli bacteria than CD14⁺⁺ monocytes, underlining their role during host defense. In addition, CD14⁺ CD16⁺ monocytes expressed higher levels of major histocompatibility complex (MHC) class II antigens (HLA-DR, -DP, and -DQ) and equal amounts of MHC class I antigens (HLA-ABC). Thus, CD14⁺ CD16⁺ cells constitute a potent phagocytosing and antigen-presenting monocyte subpopulation, which is expanded during acute and chronic infections commonly observed in chronic HD patients.

Peripheral blood monocytes are members of the mononuclear phagocytic system, which plays a central role in immunoregulation and host defense against immunopathogenic organisms (7). Monocytes are activated through molecular signals provided by structures of the infective organisms (8, 27, 28, 34, 35) or inflammatory mediators and chemotactic factors released by other cells during the infective challenge (22, 44, 47). However, blood monocytes represent a heterogeneous cell population and can be distinguished by variations in morphology (38, 58), membrane antigen expression (39), and release of inflammatory mediators (12, 25, 41).While the lipopolysaccharide (LPS) receptor antigen CD14 is expressed by nearly all circulating peripheral blood monocytes, monocytes differ markedly in cell surface CD14 density as well as in the expression of immunoglobulin Fcγ receptors (53, 67). The majority of monocytes strongly positive for CD14 (CD14⁺⁺) express Fcγ receptor I (CD64) and Fcγ receptor II (CD32) and are negative for Fcγ receptor III (CD16) (18). Only a small population was identified by the absence of Fcγ receptors (63). Nevertheless, a subset of monocytes characterized by low-level expression of CD14 and expression of the CD16 antigen has also been described (40). In healthy subjects these CD14⁺ CD16⁺ cells account for about 10% of all monocytes and are thought to be more mature cells than the regular CD14⁺⁺ monocytes, as they exhibit features of tissue macrophages (66). In various infectious or inflammatory diseases such as AIDS and asthma the CD14⁺ CD16⁺ monocyte subpopulation is markedly expanded (36, 43, 50). A more than 10-fold increase of these cells during septicemia was demonstrated, and CD14⁺ CD16⁺ cells become the predominant type of monocytes in some septic patients (14).Patients with end-stage renal failure undergoing chronic hemodialysis (HD) show an impaired immune response (10) with a high prevalence of infectious complications (17). Most of these infections are of bacterial origin, representing a major cause of morbidity and mortality in chronic HD patients (24). Furthermore, acute or chronic inflammatory processes, among them pneumonia and vascular access site infections, are common hazards in uremic patients undergoing chronic regular HD. Despite some data on the functional abnormalities of polymorphonuclear leukocytes in uremia (19), little information exists on the level of monocytes and their subsets in maintenance dialysis patients.In an effort to further understand the importance of the distinct monocyte population expressing Fcγ receptor type III, we determined the levels of these cells in patients with end-stage renal failure undergoing chronic HD. This allowed the level of CD14⁺ CD16⁺ cells to be compared to that of CD14⁺⁺ cells and the total monocyte count in whole blood. To investigate the proinflammatory role of CD14⁺ CD16⁺ monocytes, stable patients as well as patients with acute or chronic signs of infections or inflammatory processes were studied. Furthermore, we analyzed cell surface HLA expression of CD14⁺ CD16⁺ monocytes by immunophenotyping and compared their phagocytic competence with that of regular CD14⁺⁺ blood monocytes.     

A ribosomal function is necessary for efficient splicing of the T4 phage thymidylate synthase intron in vivo

Splicing of the group I intron of the T4 thymidylate synthase (td) gene was uncoupled from translation by introducing stop codons in the upstream exon. This resulted in severe splicing deficiency in vivo. Overexpression of a UGA suppressor tRNA partially rescued splicing, suggesting that this in vitro self-splicing intron requires translation for splicing in vivo. Inhibition of translation by the antibiotics chloramphenicol and spectinomycin also resulted in splicing deficiency. Ribosomal protein S12, a protein with RNA chaperone activity, and CYT-18, a protein that stabilizes the three-dimensional structure of group I introns, efficiently rescued the stop codon mutants. We identified a region in the upstream exon that interferes with splicing. Point mutations in this region efficiently alleviate the effect of a nonsense codon. We infer from these results that the ribosome acts as an RNA chaperone to facilitate proper folding of the intron.     

Frequency of Measles Virus-Specific CD4+ and CD8+ T Cells in Subjects Seronegative or Highly Seropositive for Measles Vaccine

The protective effect of measles immunization is due to humoral and cell-mediated immune responses. Little is known about cell-mediated immunity (CMI) to measles vaccine virus, the relative contribution of CD4⁺ and CD8⁺ T cells to variability in such immune responses, and the immunologic longevity of the CMI after measles vaccination in humans. Our study characterizes cellular immune response in subjects seronegative or highly seropositive for measles vaccine immunoglobulin G-specific antibody, aged 15 to 25 years, previously immunized with two doses of measles-mumps-rubella II vaccine. We evaluated the ability of subjects to respond to measles vaccine virus by measuring measles virus-specific T-cell proliferation. We examined the frequencies of measles virus-specific memory Th1 and Th2 cells by an ELISPOT assay. Our results demonstrated that proliferation of T cells in seronegative subjects was significantly lower than that for highly seropositive subjects (P = 0.003). Gamma interferon (IFN-γ) secretion predominated over interleukin 4 (IL-4) secretion in response to measles virus in both groups. The median frequency of measles virus-reactive CD8⁺ T cells secreting IFN-γ was 0.09% in seronegative subjects and 0.43% in highly seropositive subjects (P = 0.04). The median frequency of CD4⁺ T cells secreting IL-4 in response to measles virus was 0.03% in seronegative subjects and 0.09% in highly seropositive subjects (P = 0.005). These data confirm the presence of measles virus-specific cellular immune responses post-measles vaccine immunization in humans. The detection of measles virus-induced IFN-γ and IL-4 production by ELISPOT can be used to identify measles virus-specific low-frequency memory T cells in subjects immunized with measles vaccine. These differences agree in directionality with the observed antibody response phenotype.     

Irs2 Inactivation Suppresses Tumor Progression in Pten+/− Mice

Mutations in the phosphatase and tensin homologue (PTEN)/phosphatidylinositol-3 kinase-α (PI3K) signaling pathway are frequently found in human cancer. In addition, Pten^+/− mice develop tumors in multiple organs because of the activation of the PI3K signaling cascade. Because activation of PI3K signaling leads to feedback inhibition of insulin receptor substrate-2 (IRS2) expression, an upstream activator of PI3K, we therefore anticipated that IRS2 expression would be low in tumors that lack PTEN. Surprisingly, however, an elevation of IRS2 was often detected in tumor samples in which PTEN levels were compromised. To determine the potential contribution of Irs2 to tumor progression, Pten^+/− mice were crossed with Irs2^+/− mice. Deletion of Irs2 did not affect the initiation of neoplasia found in Pten^+/− mice but suppressed cancer cell growth, proliferation, and invasion through the basement membrane. Deletion of Irs2 also attenuated the expression of Myc in prostatic intraepithelial neoplasia in Pten^+/− mice. In addition, the expression levels of IRS2 and MYC were highly correlated in human prostate cancer, and IRS2 could stimulate MYC expression in cultured cells. Our findings provide evidence that the PI3K-activating adaptor Irs2 contributes to tumor progression in Pten^+/− mice by stimulating both Myc and DNA synthesis.     

Processing of Mycobacterium tuberculosis Bacilli by Human Monocytes for CD4+ αβ and γδ T Cells: Role of Particulate Antigen

Mycobacterium tuberculosis readily activates both CD4⁺ and Vδ2⁺ γδ T cells. Despite similarity in function, these T-cell subsets differ in the antigens they recognize and the manners in which these antigens are presented by M. tuberculosis-infected monocytes. We investigated mechanisms of antigen processing of M. tuberculosis antigens to human CD4 and γδ T cells by monocytes. Initial uptake of M. tuberculosis bacilli and subsequent processing were required for efficient presentation not only to CD4 T cells but also to Vδ2⁺ γδ T cells. For γδ T cells, recognition of M. tuberculosis-infected monocytes was dependent on Vδ2⁺ T-cell-receptor expression. Recognition of M. tuberculosis antigens by CD4⁺ T cells was restricted by the class II major histocompatibility complex molecule HLA-DR. Processing of M. tuberculosis bacilli for Vδ2⁺ γδ T cells was inhibitable by Brefeldin A, whereas processing of soluble mycobacterial antigens for γδ T cells was not sensitive to Brefeldin A. Processing of M. tuberculosis bacilli for CD4⁺ T cells was unaffected by Brefeldin A. Lysosomotropic agents such as chloroquine and ammonium chloride did not affect the processing of M. tuberculosis bacilli for CD4⁺ and γδ T cells. In contrast, both inhibitors blocked processing of soluble mycobacterial antigens for CD4⁺ T cells. Chloroquine and ammonium chloride insensitivity of processing of M. tuberculosis bacilli was not dependent on the viability of the bacteria, since processing of both formaldehyde-fixed dead bacteria and mycobacterial antigens covalently coupled to latex beads was chloroquine insensitive. Thus, the manner in which mycobacterial antigens were taken up by monocytes (particulate versus soluble) influenced the antigen processing pathway for CD4⁺ and γδ T cells.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is spread readily from person to person by inhalation of aerosolized mycobacteria (8). A hallmark of M. tuberculosis infection is the ability of most healthy individuals to control the infection by mounting an acquired immune response, in which antigen-specific T cells and mononuclear phagocytes arrest the growth of M. tuberculosis bacilli and maintain control over dormant bacilli within granulomas (reviewed in reference 25). This protective cellular immune response results in conversion of the tuberculin skin test from negative to positive and probably in increased resistance to reinfection with tubercle bacilli.CD4⁺ αβ-T-cell-receptor (αβ TCR)-bearing T cells (CD4⁺ T cells) are readily activated by mycobacterial antigens and have a dominant role in the protective immune response to M. tuberculosis in humans (2, 34). These CD4⁺ T cells not only secrete cytokines but also serve directly as cytotoxic effector cells against M. tuberculosis-infected macrophages (6). In addition to CD4⁺ T cells, M. tuberculosis antigens activate other human T-cell subsets such as γδ TCR⁺ T cells (γδ T cells) (15, 16, 18). Vδ2⁺ and Vγ9⁺ γδ T cells are particularly responsive to live M. tuberculosis (15). A role for both γδ and CD4⁺ T cells in protective immunity to acute M. tuberculosis infection has been demonstrated in murine models (20, 21, 26, 27). A recent study of humans suggests that Vγ9⁺ and Vδ2⁺ γδ T-cell numbers and function are reduced in tuberculosis patients (23).Functional comparisons of human CD4⁺ and γδ T-cell responses of healthy tuberculin-positive persons demonstrate that both T-cell subsets have similar cytotoxic effector functions for M. tuberculosis-infected monocytes and produce large amounts of gamma interferon (IFN-γ), with γδ T cells being slightly more efficient producers of IFN-γ than CD4⁺ T cells (37). Despite similarities in function, these two T-cell subsets differ in the mycobacterial antigens recognized by their TCRs and the manners in which antigens are presented to them by M. tuberculosis-infected mononuclear phagocytes. CD4⁺ T cells recognize a wide diversity of mycobacterial peptides in the context of class II major histocompatibility complex (MHC) molecules, which include secreted as well as somatic antigens (6, 13, 33, 37). In contrast, Vγ9⁺ and Vδ2⁺ γδ T cells, the dominant γδ TCR subsets activated by M. tuberculosis, recognize mycobacterial antigens in a non-MHC-restricted manner and the repertoire of antigens includes small phosphate-containing antigens such as TUBag’s (5, 9, 19, 22, 29, 36).Both blood monocytes and alveolar macrophages infected with M. tuberculosis are efficient antigen-presenting cells for mycobacterial antigen-specific CD4⁺ and γδ T cells (1, 5). However, little is known about how M. tuberculosis-infected mononuclear phagocytes process antigens for these two T-cell subsets. M. tuberculosis bacilli are taken up by mononuclear phagocytes through a variety of surface receptors, including complement receptor 4, mannose receptor, and complement receptor 3 (17, 31, 32). Within mononuclear phagocytes, the mycobacteria reside within phagosomes and modulate the phagosome by preventing fusion with acidic lysosomal compartments (7). Although the vacuolar membranes surrounding the phagosome acquire endosomal markers, the vesicular proton ATPase is actively excluded, resulting in an elevated pH of 6.3 to 6.5 compared to the normal lysosomal pH of 4.5 (7, 35). The elevated pH in the phagosome does not appear to inhibit the ability of mycobacterial antigens to be processed and presented to CD4⁺ and Vδ2⁺ γδ T cells. This study was undertaken to gain insight into the mechanisms used by monocytes infected with live M. tuberculosis bacilli to process mycobacterial antigens for presentation to both CD4⁺ and γδ T cells.     

Expansion of a Novel Pulmonary CD3− CD4+ CD8+ Cell Population in Mice during Chlamydia pneumoniae Infection

A new pulmonary T-cell-like lymphocyte population with the phenotype CD3⁻ CD4⁺ CD8⁺ was discovered in mice. CD4⁺ CD8⁺ but CD3⁺ cells among murine intestinal intraepithelial lymphocytes have previously been described. We describe herein a dramatic expansion of the CD3⁻ CD4⁺ CD8⁺ cell population in response to experimental respiratory infection. After intranasal Chlamydia pneumoniae infection, CD4⁺ CD8⁺ cells became transiently the dominant lymphocyte type (maximum of 87% of all lymphocytes) in the lungs of NIH/S mice but remained virtually undetectable in spleen and blood. The enrichment of these cells was not a C. pneumoniae-specific event, since infection of NIH/S mice with influenza A virus also resulted in an increase in the number of CD4⁺ CD8⁺ cells (maximum of 42% of all lymphocytes). In addition to outbred NIH/S mice, two other mouse strains were studied: BALB/c (H-2^d) and C57BL/6 (H-2^b). C. pneumoniae-infected BALB/c mice responded with an intermediate increase in the number of CD4⁺ CD8⁺ cells in lungs, whereas C57BL/6 mice did not respond. The double-positive CD4⁺ CD8⁺ cells lacked a major part of the T-cell receptor complex, being both CD3⁻ and TCR αβ⁻. However, when they were stimulated in vitro with a T-cell mitogen, they responded by proliferation but did not secrete gamma interferon. The dramatic expansion of this cell population at the infection site suggests an active role for them in respiratory infection, but the specification of this requires further study.     

Immunophenotypic Analysis of the TCR-Vβ Repertoire in 98 Persistent Expansions of CD3+/TCR-αβ+ Large Granular Lymphocytes : Utility in Assessing Clonality and Insights into the Pathogenesis of the Disease

At present, a major challenge in the initial diagnosis of leukemia of large granular lymphocytes (LGLs) is to establish the clonal nature of the expanded population. In the present study we have analyzed by flow cytometry immunophenotyping the TCR-Vbeta repertoire of 98 consecutive cases of persistent expansions of CD4(+) or CD8(+bright) CD3(+)/TCR-alphabeta(+) LGLs and compared the results with those obtained in molecular studies of TCR-beta gene rearrangements. Fifty-eight cases were considered to be monoclonal in molecular studies whereas in the remaining 40 cases there was no evidence for monoclonality (11 cases were considered oligoclonal and 29 polyclonal). The TCR-Vbeta repertoire was biased to the preferential use of one or more TCR-Vbeta families in 96% of cases, a total of 124 TCR-Vbeta expansions being diagnosed: one TCR-Vbeta expansion in 71 cases and two or more TCR-Vbeta expansions in 23 cases. The highest TCR-Vbeta expansion observed in each case was higher among monoclonal (74 +/- 19%) as compared to nonmonoclonal cases (24 +/- 14%) (P = 0.001), as did the fraction of LGLs that exhibited a TCR-Vbeta-restricted pattern (86 +/- 16% and 42 +/- 23%, respectively; P = 0.0001); by contrast, the proportion of cases displaying more than one TCR-Vbeta expansion was higher in the latter group: 7% versus 48%, respectively (P = 0.001). Results obtained in oligoclonal cases were intermediate between those obtained in polyclonal and monoclonal cases and similar results were observed for CD4(+) as for CD8(+bright) T-cell expansions. TCR-Vbeta families expressed in CD8(+bright) T-cell-LGL proliferations showed a pattern of distribution that mimics the frequency at which the individual TCR-Vbeta families are represented in normal peripheral blood T cells. Assuming that a given proliferation of LGLs is monoclonal whenever there is an expansion of a given TCR-Vbeta family of at least 40% of the total CD4(+) or CD8(+bright) T-cell compartment, we were able to predict clonality with a sensitivity of 93% and a specificity of 80%. By increasing the cut-off value to 60%, sensitivity and specificity were of 81% and 100%. In summary, our results suggest that flow cytometry immunophenotypic analysis of the TCR-Vbeta repertoire is a powerful screening tool for the assessment of T-cell clonality in persistent expansions of TCR-alphabeta(+) LGLs.     

Increased Foxp3+ CD4+ Regulatory T Cells with Intact Suppressive Activity but Altered Cellular Localization in Murine Lupus

Foxp3⁺ CD4⁺ regulatory T (T_reg) cells play a pivotal role in the maintenance of dominant self tolerance. Understanding how the failures of immune control by T_reg cells are involved in autoimmune diseases is important for the development of effective immunotherapies. In the present study, we analyzed the characteristics of endogenous T_reg cells in (NZB × NZW) F1 (BWF1) mice, a murine model of systemic lupus erythematosus. Unexpectedly, T_reg number and frequency in aged BWF1 mice with developing lupus nephritis were increased, not decreased, and in vitro suppressive activity in lymphoid organs was intact. In addition, T_reg cells trafficked to target organs because cells were present in the kidney and lung. T_reg cells of aged BWF1 mice exhibited altered localization within lymph organs, however, and an altered phenotype, with higher expression levels of chemokine receptors and activation markers, suggesting a highly activated cellular state. Notably, the expression levels of co-stimulatory molecules were also markedly enhanced in the T_reg cells of aged BWF1 mice. Furthermore, T_reg cells of BWF1 mice did not show any suppressive effects on antibody production in vitro. Taken together, we conclude that T_reg cells in BWF1 mice are not predisposed to functional incompetence but rather are present in a highly activated state.     

Low Relative Frequencies of CD26+ CD4+ Cells in Long-Term Nonprogressing Human Immunodeficiency Virus Type 1-Infected Subjects

A broad antibody panel was used for immunophenotyping of human immunodeficiency virus type 1 (HIV-1)-infected patients who were long-term nonprogressors (LTNP). The LTNP were compared with patients in the early phase of infection and patients who had progressed to advanced immunodeficiency. Changes in CD8⁺ subset distribution were observed mainly at acquisition of HIV-1 infection, whereas CD4⁺ subset changes appeared during progression of HIV-1 infection. The decreasing levels of CD4⁺ cells were characterized by an increasing frequency of cells expressing the activation markers HLA-Dr and CD45RO but not the CD28 surface antigen. The LTNP exhibited significant changes compared to HIV-negative patients in almost all markers. Compared to patients in the early phase of infection, the only difference was a relatively lower frequency of CD4⁺ cells expressing CD26 among the LTNP. The results show that HIV-1-infected persons who have no signs of immunodeficiency despite many years of infection have an immunophenotypic pattern that is substantially different from that of noninfected persons. Despite the long duration of infection, the LTNP exhibit a pattern similar to that of newly infected persons, with the exception of lower expression of CD26 on CD4⁺ cells.     

CCR1+/CCR5+ Mononuclear Phagocytes Accumulate in the Central Nervous System of Patients with Multiple Sclerosis

Mononuclear phagocytes (monocytes, macrophages, and microglia) are considered central to multiple sclerosis (MS) pathogenesis. Molecular cues that mediate mononuclear phagocyte accumulation and activation in the central nervous system (CNS) of MS patients may include chemokines RANTES/CCL5 and macrophage inflammatory protein-1alpha/CCL3. We analyzed expression of CCR1 and CCR5, the monocyte receptors for these chemokines, on circulating and cerebrospinal fluid CD14+ cells, and in MS brain lesions. Approximately 70% of cerebrospinal fluid monocytes were CCR1+/CCR5+, regardless of the presence of CNS pathology, compared to less than 20% of circulating monocytes. In active MS lesions CCR1+/CCR5+ monocytes were found in perivascular cell cuffs and at the demyelinating edges of evolving lesions. Mononuclear phagocytes in early demyelinating stages comprised CCR1+/CCR5+ hematogenous monocytes and CCR1-/CCR5- resident microglial cells. In later stages, phagocytic macrophages were uniformly CCR1-/CCR5+. Cultured in vitro, adherent monocytes/macrophages up-regulated CCR5 and down-regulated CCR1 expression, compared to freshly-isolated monocytes. Taken together, these findings suggest that monocytes competent to enter the CNS compartment derive from a minority CCR1+/CCR5+ population in the circulating pool. In the presence of ligand, these cells will be retained in the CNS. During further activation in lesions, infiltrating monocytes down-regulate CCR1 but not CCR5, whereas microglia up-regulate CCR5.     

HLA-B*35-Restricted CD8+-T-Cell Epitope in Mycobacterium tuberculosis Rv2903c

Few human CD8(+) T-cell epitopes in mycobacterial antigens have been described to date. Here we have identified a novel HLA-B*35-restricted CD8(+) T-cell epitope in Mycobacterium tuberculosis Rv2903c based on a reverse immunogenetics approach. Peptide-specific CD8 T cells were able to kill M. tuberculosis-infected macrophages and produce gamma interferon and tumor necrosis factor alpha.     

CD34+ Cord Blood Cell-Transplanted Rag2−/− γc−/− Mice as a Model for Epstein-Barr Virus Infection

Recent studies suggest that Epstein-Barr virus (EBV) can infect naïve B cells, driving them to differentiate into resting memory B cells via the germinal center reaction. This hypothesis has been inferred from parallels with the biology of normal B cells but has never been proven experimentally. Rag2^−/− γ_c^−/− mice that were transplanted with human CD34⁺ cord blood cells as newborns were recently shown to develop human B, T, and dendritic cells, constituting lymphoid organs in situ. Here we used this model to better define the strategy of EBV infection of human B cells in vivo and to compare this model system with different conditions of EBV infection in humans. Our results support the model of EBV persistence in vivo in cases that were characterized by follicular hyperplasia and a relatively normal CD4⁺ and CD8⁺ T-cell distribution. Intriguingly, in cases that were characterized by nodular and diffuse proliferation with a preponderance of CD8⁺ T cells, similar to infectious mononucleosis, EBV still infects naïve B cells but also induces clonal expansion and ongoing somatic mutations without germinal center reactions. Our results reveal different strategies of EBV infection in B cells that possibly result from variations in the host immune response. Future experiments might allow understanding of the mechanisms responsible for persistent EBV infection and provide targets for more highly tailored therapeutic interventions.     

CD8+-T-Cell Immunity against Toxoplasma gondii Can Be Induced but Not Maintained in Mice Lacking Conventional CD4+ T Cells

T-cell immunity is critical for survival of hosts infected with Toxoplasma gondii. Among the cells in the T-cell population, CD8(+) T cells are considered the major effector cells against this parasite. It is believed that CD4(+) T cells may be crucial for induction of the CD8(+)-T-cell response against T. gondii. In the present study, CD4(-/-) mice were used to evaluate the role of conventional CD4(+) T cells in the immune response against T. gondii infection. CD4(-/-) mice infected with T. gondii exhibited lower gamma interferon (IFN-gamma) messages in the majority of their tissues. As a result, mortality due to a hyperinflammatory response was prevented in these animals. Interestingly, T. gondii infection induced a normal antigen-specific CD8(+)-T-cell immune response in CD4(-/-) mice. No difference in generation of precursor cytotoxic T lymphocytes (pCTL) or in IFN-gamma production by the CD8(+)-T-cell populations from the knockout and wild-type animals was observed. However, the mutant mice were not able to sustain CD8(+)-T-cell immunity. At 180 days after infection, the CD8(+)-T-cell response in the knockout mice was depressed, as determined by pCTL and IFN-gamma assays. Loss of CD8(+)-T-cell immunity at this time was confirmed by adoptive transfer experiments. Purified CD8(+) T cells from CD4(-/-) donors that had been immunized 180 days earlier failed to protect the recipient mice against a lethal infection. Our study demonstrated that although CD8(+)-T-cell immunity can be induced in the absence of conventional CD4(+) T cells, it cannot be maintained without such cells.     

Influence of angiotensin II, α- and β-adrenoceptors on peripheral noradrenergic neurotransmission in canine gracilis muscle in vivo

Interactions between angiotensin II and adrenoceptor-mediated effects on peripheral sympathetic neurotransmission were investigated in constant flow blood-perfused canine gracilis muscle in situ, without and with pretreatment by non-competitive α- adrenoceptor blockade. Angiotensin converting enzyme (ACE)-inhibition by benazeprilat increased nerve stimulation (2 Hz, 4 min)-evoked noradrenaline (NA) overflow (+21 ± 5 yo) with α- adrenoceptors intact, but reduced NA overflow (– 18 ± 6%) when α-adrenoceptors were blocked. Vasoconstrictor responses were slightly reduced by benazeprilat. Subsequent infusion of angiotensin II (Ang 11, 20 and 500 ng kg_-1 min_-1 i.v., raising arterial concentrations from 0.6 ± 0.2 PM to 1390 ± 240 and 25 110 ± 3980 PM, respectively) failed to increase NA overflow or to enhance stimulation-evoked vasoconstriction. Adrenaline (0.4 nmol kg_-1 min_-1 i.v.) did not change evoked NA overflow before or after benazeprilat, either with or without α-adrenoceptor blockade, despite high concentrations (± 10 nM) in arterial plasma. Following benazeprilat, propranolol reduced NA overflow (–24 ± 3 yo) only if the α-adrenoceptors were blocked. In conclusion, benazeprilat reduced evoked NA overflow in the presence of α- adrenoceptor blockade to a similar degree as previously shown in the presence of neuronal uptake inhibition in this model. However, contrasting to our previous findings, benazeprilat enhanced NA overflow and reduced the post-junctional response to nerve stimulation in the absence of α-adrenoceptor blockade. This could be related to bradykinin accumulation during ACE-inhibition, in addition to the reduction of Ang II generation. Our data are not compatible with facilitation of NA release by circulating Ang II even at pharmacological dose levels. Although activation of prejunctional β-adrenoceptors may facilitate evoked NA overflow in this model, circulating adrenaline is ineffective under physiological conditions even after α-adrenoceptor blockade. Also, β-adrenoceptor-mediated prejunctional effects do not seem to involve Ang II in canine skeletal muscle in vivo.     

Identification of Amino Acid Residues of the T-Cell Epitope of Mycobacterium tuberculosis α Antigen Critical for Vβ11+ Th1 Cells

Stimulation of Mycobacterium tuberculosis-primed lymph node cells from C57BL/6 mice with alpha antigen (also known as antigen 85B and MPT59) induced cell proliferation, production of interleukin 2 and gamma interferon, and expansion of Vbeta11(+) CD4(+) T cells in conjunction with antigen-presenting cells in an I-A(b)-restricted manner. Using a series of 15-amino-acid peptides that overlapped each other by 5 amino acids and spanned the mature alpha antigen, we identified the antigenic epitope for alpha antigen-specific Vbeta11(+) Th1 cells. That peptide (peptide-25), which corresponds to amino acid residues 240 to 254 of alpha antigen, contains a motif that is conserved in I-A(b) and requires processing by antigen-presenting cells. Using peptide-25-reactive Vbeta11(+) T-cell clones and substituted peptide-25 mutants, we determined which amino acid residues within peptide-25 were critical for T-cell receptor (TCR) recognition. Our results showed that the amino acid residues at positions 245, 246, 248, 250, and 251 are important for recognition of TCRVbeta11 and that residues at positions 244, 247, 249, and 252 are I-A(b) contact residues. We also observed that active immunization of C57BL/6 mice with peptide-25 can lead to decreased bacterial load in the lungs of M. tuberculosis H37Rv-infected mice. These results should provide us with a useful tool for delineating the regulation of Vbeta11(+) Th1-cell development during M. tuberculosis infection and for developing a vaccine inducing a Th1-dominant immune response.     

Primary Role for CD4+ T Lymphocytes in Recovery from Oropharyngeal Candidiasis

Oropharyngeal candidiasis is associated with defects in cell-mediated immunity and is commonly seen in human immunodeficiency virus positive individuals and AIDS patients. A model for oral candidiasis in T-cell-deficient BALB/c and CBA/CaH nu/nu mice was established. After inoculation with 10(8) Candida albicans yeasts, these mice displayed increased levels of oral colonization compared to euthymic control mice and developed a chronic oropharyngeal infection. Histopathological examination of nu/nu oral tissues revealed extensive hyphae penetrating the epithelium, with polymorphonuclear leukocyte microabscess formation. Adoptive transfer of either naive or immune lymphocytes into immunodeficient mice resulted in the recovery of these animals from the oral infection. Reconstitution of immunodeficient mice with naive CD4(+) but not CD8(+) T cells significantly decreased oral colonization compared to controls. Interleukin-12 and gamma interferon were detected in the draining lymph nodes of immunodeficient mice following reconstitution with naive lymphocytes. This study demonstrates the direct requirement for T lymphocytes in recovery from oral candidiasis and suggests that this is associated with the production of cytokines by CD4(+) T helper cells.