Aged mice display an altered pulmonary host response to Francisella tularensis live vaccine strain (LVS) infections.

Aging is a complex phenomenon that has been shown to affect many organ systems including the innate and adaptive immune systems. The current study was designed to examine the potential effect of immunosenescence on the pulmonary immune response using a Francisella tularensis live vaccine strain (LVS) inhalation infection model. F. tularensis is a Gram-negative intracellular pathogen that can cause a severe pneumonia. In this study both young (8-12 week old) and aged (20-24 month old) mice were infected intranasally with LVS. Lung tissues from young and aged mice were used to assess pathology, recruitment of immune cell types and cytokine expression levels at various times post infection. Bacterial burdens were also assessed. Interestingly, the lungs of aged animals harbored fewer organisms at early time points of infection (day 1, day 3) compared with their younger counterparts. In addition, only aged animals displayed small perivascular aggregates at these early time points that appeared mostly mononuclear in nature. However, the kinetics of infiltrating polymorphonuclear neutrophils (PMNs) and increased cytokine levels measured in the bronchial alveolar lavage fluid (BALF) were delayed in infected aged animals relative to young infected animals with neutrophils appearing at day 5 post infection (PI) in the aged animals as opposed to day 3 PI in the young infected animals. Also evident were alterations in the ratios of mononuclear to PMNs at distinct post infection times. The above evidence indicates that aged mice elicit an altered immune response in the lung to respiratory F. tularensis LVS infections compared to their younger counterparts.

The human nervous tissue in proximity to granulomatous lesions induced by Taenia solium metacestodes displays an active response.

In neurocysticercosis, the nervous tissue surrounding the brain lesion is affected as a consequence of the local immune response induced by a Taenia solium metacestode. In this study, a histological and immunohistochemical analysis of five brain specimens from patients with neurocysticercosis revealed a proinflammatory activity reflected by an apparently altered blood-brain barrier permeability, secretion of pro-inflammatory cytokines, and up-regulation of molecules associated with antigen presentation. There were also anti-inflammatory cytokines, as well as an active wound-healing process reflected by angiogenesis, collagen deposition and glial scar formation. This immune response displayed by the nervous tissue adjacent to chronic neurocysticercosis lesions appeared to be contributing to the local tissue damage, and hence, may be fundamental in the pathology of NCC.

Leishmania donovani: evolution and architecture of the splenic cellular immune response related to control of infection.

Infection with the protozoan Leishmania donovani in humans is usually subclinical. Parasites probably persist for the life of the host and the low-level infection is controlled by the cellular immune response. To better understand the mechanisms related to the control of infection, we studied the evolution and architecture of the splenic cellular immune response in a murine model that is most representative of human subclinical infection. Following systemic inoculation with L. donovani, the parasites were primarily localized to the macrophage-rich splenic red pulp. There was an initial increase in the numbers of T cells and dendritic cells in the periarteriolar lymphoid sheath and marginal zone, but the red pulp (where parasitized macrophages were prominent) remained free of these cells until later in the course of infection. Thus, T cells did not colocalize with parasitized red pulp macrophages until later in the course of infection. Early in the course of infection, IL-10 production within the marginal zone and TGF-beta production by cells in the red pulp were prominent. These macrophage-inhibitory cytokines may contribute to the establishment of the infection and early parasite replication. By day 28 of infection, when the visceral parasite burden began to decline, the number of IL-10-producing spleen cells was back to the baseline level, but IFN-gamma production was higher and the number of IL-12-producing cells was increased dramatically. At this time T cells and dendritic cells had moved out of the lymphoid follicle and marginal zone into the red pulp where the parasites were located. These findings therefore suggest that control of infection is associated with IFN-gamma and IL-12 production and migration of T cells and dendritic cells to the site of chronic parasitism.

Analysis of the peripheral immune response in patients with neurocysticercosis: evidence for T cell reactivity to parasite glycoprotein and vesicular fluid antigens.

In neurocysticercosis (NCC), it is thought that the long-term survival of the parasite within the human brain is due in part to the ability of the cestode to suppress the local immune response. When the parasite dies, the immunosuppression is apparently lost and a strong local inflammatory response then develops. In contrast, little is known about the immunologic response that may occur in the peripheral immune system of these patients. In this study, the status of the peripheral (extracerebral) cellular and humoral response was evaluated in patients with a history of NCC. The in vitro proliferation of peripheral blood mononuclear cells to mitogens and foreign antigens was similar in patients and controls. Importantly, a substantive response was elicited by two Taenia solium metacestode antigens. In addition, 8 of 10 patients had a detectable humoral response to the antigenic glycoproteins of the cestode. Considering both the cellular and humoral response, all of the patients with NCC presented an active peripheral immunity.

Brain granulomas in neurocysticercosis patients are associated with a Th1 and Th2 profile.

Neurocysticercosis (NCC) is a common central nervous system (CNS) infection caused by Taenia solium metacestodes. Despite the well-documented importance of the granulomatous response in the pathogenesis of this infection, there is limited information about the types of cells and cytokines involved. In fact, there has been limited characterization of human brain granulomas with any infectious agent. In the present study a detailed histological and immunohistochemical analysis of the immune response was performed on eight craniotomy specimens where a granuloma surrounded each T. solium metacestode. The results indicated that in all the specimens there was a dying parasite surrounded by a mature granuloma with associated fibrosis, angiogenesis, and an inflammatory infiltrate. The most abundant cell types were plasma cells, B and T lymphocytes, macrophages, and mast cells. Th1 cytokines were prevalent and included gamma interferon, interleukin-18 (IL-18), and the immunosuppressive, fibrosis-promoting cytokine transforming growth factor beta. The Th2 cytokines IL-4, IL-13, and IL-10 were also present. These observations indicate that a chronic immune response is elicited in the CNS environment with multiple cell types that together secrete inflammatory and anti-inflammatory cytokines. In addition, both collagen type I and type III deposits were evident and could contribute to irreversible nervous tissue damage in NCC patients.

T cell expansions in lymph nodes and peripheral blood in HIV-1-infected individuals: effect of antiretroviral therapy.

OBJECTIVE: To evaluate dynamics in CD8 T cell expansions during highly active antiretroviral therapy (HAART). DESIGN: Various T cell subsets were isolated from blood and lymph nodes and analysed for T cell receptor (TCR) diversity. METHODS: TCR complementarity determining region 3 (CDR3) spectratyping and single-strand conformation polymorphism (SSCP) analyses were performed in combination with sequencing to assess clonality of the subsets. RESULTS: Strongly skewed CDR3 patterns in total CD8 cells and the CD8 subsets CD45RO+CD27+ and CD45RO-CD27+ showed substantial dynamics in dominant CDR3 sizes, resulting in relative improvement of CDR3 size diversity in the first months of therapy. During sustained treatment, TCR diversity changed only moderately. SSCP profiles confirmed oligoclonality of TCR CDR3 perturbations. Various dominant CDR3 sizes for CD4 and CD8 T cells present in lymph nodes, but not in peripheral blood mononuclear cells, before the start of therapy emerged in peripheral blood early during therapy. CONCLUSIONS: HAART induces substantial changes in CD8 TCR diversity, eventually resulting in improvement of the repertoire. Clonal expansions observed in lymph nodes before therapy were observed in peripheral blood after therapy, suggesting that recirculation of CD4 and CD8 T cells from lymph nodes contributes to the early T cell repopulation. Decreased immune activation and possibly naive T cell regeneration subsequently decreased clonal expansions and perturbations in the CD8 TCR repertoire.

The role of N-linked carbohydrates in the antigenicity of Taenia solium metacestode glycoproteins of 12, 16 and 18 kD.

The glycoproteins of 12-28 kD from Taenia solium metacestodes provide a high specificity and sensitivity for the serological diagnosis of the central nervous system infection, neurocysticercosis. Their widespread use as antigens for routine serological assays will require their production in large and reproducible amounts. Prior to determining the ideal strategy to produce these antigens at a large scale, it is important to determine the contribution of the carbohydrates to the antigenicity of these molecules, given the uncertainty of reproducing saccharidic epitopes in recombinant expression systems. In this study we examined this issue. The chemical oxidation of the carbohydrates of the 12-28 kD glycoproteins with sodium metaperiodate, reduced the antigenicity of the molecules to variable extents, with the more notable changes being detected for the 18 and 28 kD antigens. This approach was complemented by purification of the 12, 16 and 18 kD antigens, followed by the enzymatic deglycosylation of their abundant N-linked oligosaccharides. Silver-stained SDS-PAGE analysis indicated that the three deglycosylated antigens now migrated as 7 kD products, suggesting a protein backbone with a similar size, but different extents of glycosylation. By Western blot, the antigenicity of these antigens was diminished. This was more notable for the 18 kD antigen, which is more heavily glycosylated than the 12 or 16 kD glycoproteins. These data suggest that the antigenicity of the glycoproteins of T. solium is due to a combination of carbohydrate and protein epitopes.

Ig heavy chain CDR3 size diversities are similar after conventional peripheral blood and ex vivo expanded hematopoietic cell transplants.

It is largely unknown whether the immune repertoire can be reconstituted successfully after high-dose chemotherapy and transplantation using ex vivo expanded hematopoietic stem cell (HSC) grafts. It is critically important for the transplant outcome that immune repertoire reconstitution progresses after ex vivo expanded HSC graft transplants at least as efficiently as that seen after conventional HSC transplants. Previously, we showed that the T cell receptor V beta (TCRVB) third complementarity determining region (CDR3) diversification after ex vivo expanded bone marrow (BM) HSC graft transplants was similar to that seen after conventional peripheral blood stem cell transplants (PBSCTs). In the present study, the CDR3 diversity of the six immunoglobulin (Ig) heavy chain variable region gene (V(H)) families was examined in five breast cancer patients who were transplanted with ex vivo expanded BM HSCs as the only source of stem cells. For comparison, 12 healthy adults and four conventional PBSCT recipients were also studied. Using both CDR3 fingerprinting and single strand conformation polymorphism (SSCP) methodologies, it is shown that the contribution of the V(H) families to the overall repertoire among healthy adults is highly variable and not always proportional to V(H) family member size. After both ex vivo expanded HSC transplants and conventional PBSCTs, the V(H) CDR3 repertoires were limited in size diversity at 6 weeks post transplant. By 6 months, however, V(H) families displayed a repertoire diversity that was as complex as that seen in healthy adults. No difference was seen between ex vivo expanded HSC graft transplant recipients and conventional PBSCT recipients in V(H) repertoire diversity. In one patient there was a follow-up analysis 12 months after ex vivo expanded graft transplant, and the diversity of the V(H) families was maintained. In all patients, the amino acid size of the CDR3 regions fell within adult limits at all time points post transplant. These results indicate that B cell repertoire regeneration after ex vivo expanded hematopoietic cell graft transplants is similar to that seen after conventional PBSCT.

Ex vivo-expanded hematopoietic cell graft recipients exhibit T cell repertoire diversity similar to that seen after conventional stem cell transplants.

The feasibility of using ex vivo-expanded hematopoietic progenitor cells to reconstitute hematopoiesis after high-dose chemotherapy is presently being examined. Early studies have shown that myeloid and erythroid hematopoiesis can be successfully reconstituted after high-dose chemotherapy and ex vivo-expanded hematopoietic cell transplantation. The lymphoid reconstitution, however, has not been addressed previously. In this study, we examined the diversity of the T cell receptor V beta chain (TCRBV) repertoires in 5 breast cancer patients who were transplanted with ex vivo-expanded bone marrow mononuclear cells as the only source of hematopoietic graft. Using the TCRBV third complementarity determining region (CDR3) fingerprinting methodology, it is shown that CD4(+) and CD8(+) T cell subsets after ex vivo-expanded hematopoietic cell graft transplants exhibit TCRBV diversities that are similar in complexity when compared to those seen after conventional autologous peripheral blood stem cell transplants (PBSCT). No apparent difference in the extent of CDR3 diversity was found between ex vivo expanded and conventional autologous PBSCT recipients when the CD4(+) and CD8(+) subsets were further separated into CD45RA(+) "naïve" and CD45RO(+) "memory" subsets. The diversity of the CD45RA(+) naïve subsets was as complex as that of the CD45RO(+) memory subsets. These results indicate that T cell repertoire diversification is not further compromised when ex vivo-expanded hematopoietic cells are used instead of autologous peripheral blood stem cells as the only source of graft.

Characterisation of the carbohydrate components of Taenia solium metacestode glycoprotein antigens.

Human neurocysticercosis is caused by Taenia solium metacestodes. It usually affects the central nervous system of humans and can be confused with other brain pathologies. The Lens culinaris-binding glycoproteins from this parasite have been shown to be ideal targets for the development of a highly specific immunoassay for the diagnosis of neurocysticercosis. In the present study we characterised the carbohydrates associated with five antigenic glycoproteins of T. solium metacestodes in the range of 12-28 kilodaltons. Lectin-affinities and enzymatic deglycosylations suggested that each of the five antigens contain various glycoforms of asparagine-linked carbohydrates of the hybrid, complex and probably high mannose type. These carbohydrates accounted for at least 30-66% of the apparent molecular mass of the glycoconjugates. In contrast, there was no evidence for the presence of O-linked carbohydrates. Lectin affinity patterns suggested that the sugars are short and truncated in their biosynthetic route, and that some contain terminal galactose moieties. Elucidating the precise structure of the carbohydrates and establishing their role in antigenicity will be essential to design strategies to produce them in large and reproducible amounts for the development of improved immunoassays.

Development of an animal model for neurocysticercosis: immune response in the central nervous system is characterized by a predominance of gamma delta T cells.

Neurocysticercosis is the most common parasitic disease of the central nervous system worldwide. It is caused by the metacestode form of the helminth Taenia solium. Study of the immune response in the human brain has been limited by the chronic progression of the disease, the influence of corticosteroid treatment, and the scarcity of patients who undergo surgical intervention. To better understand the immune response and associated pathology in neurocysticercosis, a mouse model was developed by intracranial infection of BALB/c mice with Mesocestoides corti, a cestode organism related to T. solium. The immune response reveals the presence of abundant inflammatory infiltrates appearing as early as 2 days postinfection in extraparenchymal regions. In contrast, infiltration of immune cells into parenchymal tissue is significantly delayed. There is a natural progression of innate (neutrophils and macrophages), early induced (NK cells and gamma delta T cells), and adaptive immune responses (alpha beta T cells and B cells) in infected mice. Gamma delta T cells are the predominant T cell population. A cell-mediated Th1 pathway of cytokine expression is evident in contrast to the previously described Th2 phenotype induced in the periphery.

Analysis of clonal diversity in mouse immunoglobulin heavy chain genes selected for size of the antigen combining site.

Size-diversity of Ig and T cell receptor antigen binding (CDR3) regions can be visualized by "CDR3 fingerprinting", and provides an estimate of B- or T-cell repertoire complexity. The method does not identify clonal diversity, however, which can only be determined by random sequencing of the CDR3s. In this study we demonstrate that a combination of fingerprinting and single strand conformation polymorphism (SSCP) analysis can be used for a rapid estimation of clonal diversity within mouse Ig antigen binding regions selected for size. This application may be useful in the analysis of clonal expansion within B- and T-cell repertoires.

Ig heavy chain third complementarity determining regions (H CDR3s) after stem cell transplantation do not resemble the developing human fetal H CDR3s in size distribution and Ig gene utilization.

Previous studies have suggested that the B-cell repertoire after stem cell transplantation resembles the developing repertoire in the fetus. Fetal and adult repertoires differ strikingly at the molecular level in Ig heavy chain third complementarity determining region (H CDR3) size distribution and Ig gene utilization. Previously, the posttransplant repertoire has not been studied fully in this regard. In this study, we analyzed H CDR3s posttransplant using CDR3 fingerprinting, single-strand conformation polymorphism (SSCP), and random sequencing. Eleven adult patients who received either autologous (n = 6) or allogeneic adult sibling (n = 5) hematopoietic stem cell transplants were studied. IgM H CDR3 repertoires demonstrated limited clonal diversity within the first 6 to 10 weeks posttransplant. By 3 to 4 months, the IgM H CDR3 repertoires were as diverse as those in healthy adults. Reconstitution of the IgM diversity correlated with the expansion of the multimember VH3 family. By contrast, the contribution of the single-member VH6 family was limited in most patients up to 6 to 9 months. No evidence was seen for greater contribution of VH6 posttransplant. IgG repertoires remained clonally restricted at all times. In all patients, H CDR3 sizes fell within adult limits. Direct nucleotide sequencing of H CDR3s showed adult-type N-nucleotide insertions and Ig gene utilization. These results indicate that the emerging repertoire posttransplant does not resemble the developing fetal repertoire at the molecular level.

Analysis of immune lesions in neurocysticercosis patients: central nervous system response to helminth appears Th1-like instead of Th2.

Neurocysticercosis (NCC) caused by the helminth Taenia solium is the most common parasitic infection of the human central nervous system (CNS) worldwide. Because clinical symptoms are associated with localized immunological responses in the brain, characterization of these responses are pivotal for understanding the pathogenesis of cysticercosis. Immunohistochemical analysis of brain specimens from several patients with cysticercosis revealed at least four types of immune responses, including: (i) an antibody response (IgM + plasma cells), (ii) a predominant NK response, (iii) an infiltrate with abundant macrophages and granulocytes, and (iv) an intense infiltrate with a predominance of macrophages and T cells. The intensity and type of immunity appeared to be associated somewhat with the parasite's viability and anatomical location. In most of the lesions, cell mediated responses were evident and proinflammatory cytokines including IL12 predominated. Moreover, IL4 was undetectable in the immune infiltrates. Thus, the CNS response to this helminth, unlike the systemic response, is predominately Th1-like.

T cell receptor repertoire diversity and clonal expansion in human neonates.

Newborn human infants, particularly those born prematurely, are susceptible to infection with a variety of microorganisms. We questioned whether limitations in the T cell repertoire contribute to the neonatal immunocompromised state. To describe developmental changes of the T cell repertoire, cDNA segments corresponding to third complementarity regions (CDR3) of human umbilical cord blood T cell receptors (TCR) from 24-41-wk gestational age were amplified with TCR family-specific probes. The resulting amplified CDRs were visualized by fingerprinting and single strand conformation polymorphism (SSCP) analysis. At 24-wk gestation there were no limitations in TCRBV family usage, and the degree of CDR3 size heterogeneity was not different from the adult. However, earlier in gestation, CDR3s were shorter for all families and gradually increased in size until term. The extent of oligoclonal expansion observed in cord blood was greater than in adult peripheral blood (p = 0.03). T cell oligoclonal expansion was greatest at 29-33-wk gestation and declined toward term. Expansions were detectable in both CD4+ and CD8+ subpopulations. Our findings indicate that the genetic mechanisms of repertoire diversification appear intact as early as 24 wk of gestation, but repertoire diversity is limited as a result of smaller CDR3 sizes. In addition, there was a developmentally regulated progression of oligoclonally expanded T cells. These differences in the TCRBV repertoire add to the body of evidence demonstrating immaturity of the neonatal immune system. However, the role that these subtle differences are likely to play in the relative immunodeficiency of the neonate remains to be determined.

Diversity of the T-cell receptor BV repertoire in HIV-1-infected patients reflects the biphasic CD4+ T-cell repopulation kinetics during highly active antiretroviral therapy.

OBJECTIVES: Highly active antiretroviral therapy (HAART) induces a decline in viral load and a biphasic increase in peripheral blood CD4+ T-cell counts in HIV-infected patients. To evaluate the effect of HAART on T-cell receptor (TCR) diversity of repopulating naive and memory CD4+ T cells, complementarity determining region 3 (CDR3) spectratyping was performed. DESIGN: For four patients treated with HAART, CD45RO+ (memory) and CD45RA+ (naive) CD4+ T cells were isolated from peripheral blood leukocyte samples obtained 1 week before, 1-2 months after, and 9-11 months after start of treatment. METHODS: CDR3 regions were amplified by TCR-BV-specific nested PCR from CD4+ T-cell subsets. CDR3 size distributions and single-strand conformation polymorphism profiles were compared as an indication for TCR diversity. RESULTS: Increasing blood CD4+ T-cell counts during the first 2 months of treatment coincided with increased perturbation of CDR3 patterns in CD4+ T-cell subsets, suggesting an early oligoclonal repopulation. At later timepoints, CDR3 size diversity increased when T-cell counts did not substantially decrease. Memory and naive CD4+ T cells generally showed comparable levels of perturbation. CONCLUSION: Diversity of the TCR repertoire reflected biphasic T-cell repopulation during HAART, compatible with initial redistribution and later CD4+ T-cell production. Sustained elevation of T-cell counts will in principle result in restoration of TCR diversity.

The role of recombination signal sequences in the preferential joining by deletion in DH-JH recombination and in the ordered rearrangement of the IgH locus.

The bias favoring deletion over inversion in DH-JH rearrangement has been known for years, but the underlying mechanism has yet to be fully defined. It has been suggested that the ratio of deletion/inversion is determined by the combined effect of two factors: (i) the relative strengths of 5' and 3' recombination signal sequences (RSS) of a DH segment, and (ii) the efficiency with which the deletional product (one joint) forms relative to the inversional product (two joints). In this study, we analyzed for the first time the effect of factor 1 alone on the biased 3' RSS utilization in DH-JH joining by using deletional plasmids in an extrachromosomal substrate V(D)J recombination assay. It was found that the 3' RSS and associated coding end (12 bp) mediate recombination more efficiently than the 5' RSS/coding end DH-JH plasmids. These results demonstrate that the effect of the RSS/coding end alone can account, at least partially, for the predominant deletion in DH-JH recombination. The potential effect of the relative strength of RSS and associated coding end on the ordered rearrangement of DH-JH followed by VH to DH-JH was also assessed. When recombination frequencies of D-->J (3' DH to J3) were compared with frequencies of V-->D (VHPJ14 to 3' DH or VHOX2 to 3' DH), it was found that V-->D joining was, if anything, more efficient than D-->J joining. Therefore, if all three segments were accessible, RSS/coding end effects would not contribute to the ordered rearrangement of the IgH locus.

Model for the neuromuscular complications of systemic lupus erythematosus.

The purpose of this study is to evaluate nerve and muscle physiology and histopathology in a murine lupus model. Muscle strength, compound muscle action potentials (distal latency and amplitude), proximal limb muscle, sciatic nerve and joint specimens were studied in MRL/lpr (lupus model) and MRL/++ (control) mice. MRL/lpr mice showed decreased muscle strength (P < 10(-6, Wilcoxon rank sum), lower compound muscle action potential mean amplitude and prolonged distal latency (P = 0.005 and 0.042. Mann-Whitney U-test), and muscle and nerve inflammation (P = 0.002 and P = 0.037, Fisher's exact test) compared with MRL/++ mice. The MRL/lpr strain evaluated in this study demonstrated muscle weakness, abnormal motor nerve conduction studies and inflammation of both muscle and nerve. These features make it an excellent model for studying the neuromuscular complications of lupus.

Frequency of B cells expressing germ-line gamma 1 transcripts upon IL-4 induction.

Murine B cells when activated with LPS and IL-4 have been shown to secrete IgG1 and IgE with corresponding H chain gene rearrangements. Using this system it has been previously demonstrated that transcription of gamma 1 and epsilon C region genes in their germ-line configuration occurs before switch recombination. Here, for the first time, the frequencies of B cells expressing germ-line gamma 1 transcripts are analyzed using in situ hybridization methodology. The results indicate that the combination of LPS and IL-4 induces a relatively low frequency of cells (5-12%) to express germ-line gamma 1 transcripts. Germ-line gamma 1 transcript-expressing cells were first detected on day 1 of culture with LPS and IL-4 and reached a maximum by days 3 to 5. The B cells that expressed germ-line gamma 1 transcripts appeared to be activated based on size and morphology. The frequency of B cells expressing germ-line gamma 1 transcripts approximated the frequency of C gamma 1+ plasma cells that appeared later in the cultures. These data may suggest a correlation between germ-line Ig transcript expression and the frequency of B cell precursors committed to the expression of a particular isotype.