Cervical Lymph Node Cells Research Articles

Characteristic features of B cells in murine cervical lymph nodes

Conclusion. B cells in cervical lymph nodes correspond to typical conventional B cells (B-2). Objective. The special status of cervical lymph nodes in relation to the oropharynx, and the need to maintain the integrity of the oropharnygeal mucosal barrier, suggest the possibility that cervical lymph node B cells located in the oropharynx may behave differently from B cells located elsewhere. In this study we examined the symmetry or lack thereof between cervical lymph node B cells and other B-cell subsets. Material and methods. We isolated B cells from murine cervical lymph node tissue and evaluated them in vitro according to several criteria. Results. We found that cervical lymph node B cells expressed typical B-cell phenotypic markers and proliferated normally in response to mitogenic stimulation. They did not spontaneously secrete immunoglobulin and, in keeping with this, did not express elevated levels of either CD138 (Syndecan-1), a marker for plasma cells, or BLIMP-1, a putative master regulator of B-cell differentiation.

A Bacterial Flagellin, Vibrio vulnificus FlaB, Has a Strong Mucosal Adjuvant Activity To Induce Protective Immunity

Flagellin, the structural component of flagellar filament in various locomotive bacteria, is the ligand for Toll-like receptor 5 (TLR5) of host cells. TLR stimulation by various pathogen-associated molecular patterns leads to activation of innate and subsequent adaptive immune responses. Therefore, TLR ligands are considered attractive adjuvant candidates in vaccine development. In this study, we show the highly potent mucosal adjuvant activity of a Vibrio vulnificus major flagellin (FlaB). Using an intranasal immunization mouse model, we observed that coadministration of the flagellin with tetanus toxoid (TT) induced significantly enhanced TT-specific immunoglobulin A (IgA) responses in both mucosal and systemic compartments and IgG responses in the systemic compartment. The mice immunized with TT plus FlaB were completely protected from systemic challenge with a 200x minimum lethal dose of tetanus toxin. Radiolabeled FlaB administered into the nasal cavity readily reached the cervical lymph nodes and systemic circulation. FlaB bound directly to human TLR5 expressed on cultured epithelial cells and consequently induced NF-kappaB and interleukin-8 activation. Intranasally administered FlaB colocalized with CD11c as patches in putative dendritic cells and caused an increase in the number of TLR5-expressing cells in cervical lymph nodes. These results indicate that flagellin would serve as an efficacious mucosal adjuvant inducing protective immune responses through TLR5 activation.

Evidence that T-Helper Type 2 Cell-Derived Cytokines and Eosinophils Contribute to Acute Rejection of Orthotopic Corneal Xenografts in Mice

Because guinea pig corneal xenografts are rejected acutely (within 16 days) in mouse eyes by a T-cell-dependent mechanism, the authors wished to determine the functional phenotype of CD4+ effector T cells. Orthotopic corneal xenotransplantation was performed from strain 13 guinea pigs to BALB/c mice. Grafted eyes were removed at specified times and examined histologically or subjected to cytokine and chemokine mRNA analysis using a multi-probe ribonuclease protection assay. Draining cervical lymph node cells were harvested at specified times and stimulated in vitro with x-irradiated strain 13 guinea pig spleen cells. Supernatants were assayed by enzyme-linked immunosorbent assay for content of interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-5, and IL-10 and cells were used for mRNA analysis. Rejected corneal xenografts were heavily infiltrated with polymorphonuclear leukocytes, the majority of which were eosinophils. These eyes contained mRNA for IL-4, IL-6, IL-10, IL-13, IL-15, and IFN-gamma. When stimulated with guinea pig spleen cells, T cells from draining cervical lymph nodes secreted primarily IL-4, IL-5, IL-10, and IFN-gamma. Eotaxin was overexpressed in eyes with rejected corneal xenografts. Acute rejection of corneal xenografts in mice is mediated by T cells that display a mixed T-helper (Th) type 2/Th1 phenotype and secrete eotaxin, an eosinophil chemoattractant. Eosinophil-dependent xenograft rejection bears similarities to immune elimination of parasites.

Immunization with dendritic cells can break immunological ignorance toward a persisting virus in the central nervous system and induce partial protection against intracerebral viral challenge.

Dendritic cells (DCs) have been used successfully to induce CD8 T cells that control virus infections and growth of tumours. The efficacy of DC-mediated immunization for the control of neurotropic Borna disease virus (BDV) in mice was evaluated. Certain strains of mice only rarely develop spontaneous neurological disease, despite massive BDV replication in the brain. Resistance to disease is due to immunological ignorance toward BDV antigen in the central nervous system. Ignorance in mice can be broken by immunization with DCs coated with TELEISSI, a peptide derived from the N protein of BDV, which represents the immunodominant cytotoxic T lymphocyte epitope in H-2(k) mice. Immunization with TELEISSI-coated DCs further induced solid protective immunity against intravenous challenge with a recombinant vaccinia virus expressing BDV-N. Interestingly, however, this immunization scheme induced only moderate protection against intracerebral challenge with BDV, suggesting that immune memory raised against a shared antigen may be sufficient to control a peripherally replicating virus, but not a highly neurotropic virus that is able to avoid activation of T cells. This difference might be due to the lack of BDV-specific CD4 T cells and/or inefficient reactivation of DC-primed, BDV-specific CD8 T cells by the locally restricted BDV infection. Thus, a successful vaccine against persistent viruses with strong neurotropism should probably induce antiviral CD8 (as well as CD4) T-cell responses and should favour the accumulation of virus-specific memory T cells in cervical lymph nodes.

Nasal delivery of antigen with the B subunit of Escherichia coli heat-labile enterotoxin augments antigen-specific T-cell clonal expansion and differentiation.

Escherichia coli heat-labile enterotoxin has unique immunogenic and adjuvant properties when administered mucosally to mice. These properties have revealed the potential for its use in the development of mucosal vaccines, an area of increasing interest. However, the inherent toxicity mediated by the A subunit precludes its widespread use. This problem has led to attempts to dissociate toxicity from adjuvant function by use of the B subunit. The ability of the B subunit of E. coli heat-labile enterotoxin (EtxB) to enhance responses against antigens coadministered intranasally is demonstrated here with the use of the DO11.10 adoptive-transfer model, in which ovalbumin (OVA)-specific adoptively transferred T cells can be monitored directly by flow cytometry. Intranasal delivery of OVA with EtxB resulted in increased T-cell proliferative and systemic antibody responses against antigens. The increased Th2 cytokine production detected following in vitro restimulation of splenocyte and cervical lymph node (CLN) cells from the immunized mice correlated with increased OVA-specific immunoglobulin G1 antibody production. Flow cytometric analysis of T cells from mice early after immunization directly revealed the ability of EtxB to support antigen-specific clonal expansion and differentiation. Furthermore, while responses were first detected in the CLNs, they rapidly progressed to the spleen, where they were further sustained. Examination of CD69 expression on dividing cells supported the notion that activation induced by the presence of antigens is not sufficient to drive T-cell differentiation. Furthermore, a lack of CD25 expression on dividing cells suggested that EtxB-mediated T-cell clonal expansion may occur without a sustained requirement for interleukin 2.

Dynamics of mast cells in lymph node following antigenic stimulation.

Dynamics of mast cells in rat cervical lymph nodes were examined using conventional histological techniques after injection of Salmonella paratyphi B-H antigen. There was no significant change in the number of mast cells at sixth hour and on the first day of stimulation compared with the controls. The number of mast cells was increased in all lymph node compartments on the second day of stimulation, which continued in the following 3 days. On the eighth day of stimulation, although the mast cell number decreased in the subcapsular area, it was still high in the paracortical area and medullary sinuses of the lymph nodes. On the second day of stimulation, the mast cell number was apparently increased in the subcapsular area than those of the other compartments. In the following days of stimulation, the highest number of mast cells was seen in the medullary sinuses. The highest paracortical mast cell number was determined on the third day of stimulation and some mast cells were observed near the high endothelial venules (HEVs). The changes of mast cell number among the lymph node compartments after antigenic stimulation support the hypothesis that the migration of mast cells occurred. This migration pattern indicates that mast cells enter the lymph node via afferent lymphatics and migrate to the lymph node compartments following antigenic stimulation.

Basic and clinical studies on quantitative analysis of lymph node micrometastasis in oral cancer

Metastasis to cervical lymph nodes (LN) is significantly associated with the outcome of patients with oral cancer. To provide a useful method for the detection of micrometastases, we analyzed 115 LNs from 10 patients with oral cancer using real-time quantitative polymerase chain reaction (PCR) based on the expression of squamous cell carcinoma antigen (SCCA) and cytokeratin 13 (CK13). The sensitivity and quantification of this method were assessed by means of limited dilution of cultured oral cancer cells and a model of cervical LN-metastasis established by inoculating green fluorescent protein (GFP)-expressing cells into the tongue of nude mice. In both investigations, a few cancer cells were detected by real-time quantitative PCR, but not by conventional reverse transcription-PCR (RT-PCR). SCCA mRNA was detected at high levels in metastatic LNs. In contrast, 26 of the 30 control cervical LNs did not express the gene at all, and the rest showed fairly low levels. Of 108 histologically metastasis-negative LNs, 19 (17.6%) expressed SCCA mRNA levels higher than the cut-off value (1.0: mean expression of control LNs + 2SD). CK13 mRNA is not a suitable marker for the real-time PCR since it was detected frequently even in the control LNs. These findings suggest that genetic diagnosis by real-time quantitative PCR based on SCCA mRNA expression may be clinically useful for detecting occult tumor cells in cervical LNs.

Diesel Exhaust-Associated Gas Components Enhance Chemokine Production by Cervical Lymph-Node Cells from Mice Immunized with Sugi Basic Proteins

Previously, we showed that exposure to diesel exhaust (DE) increased inflammatory cells in the airway and cytokine production from local lymph-node cells after antigen stimulation. To clarify the role of particle-free diesel gas components in induction of allergic inflammation, we compared the effect of DE and gas components on pollen-antigen-stimulated chemokine production by cervical lymph nodes (CLN) cells in BALB/c mice. Groups of mice were exposed to 0 (control), 1.0 mg diesel exhaust particles (DEP)/m3 (DE), or filtered 1.0 mg DEP/m3 DE (gas) for 12 h daily for 5 wk. Each group of mice was injected intraperitoneally with sugi basic protein (SBP), a major allergen of Japanese cedar pollen, immediately before their exposure to DE or gas. On days 14 and 35, each mouse received an additional SBP intranasally. Exposure to DE or gas did not affect the lymphocyte subpopulations of CLN. Culture supernatants of CLN cells from DE-exposed, SBP-immunized mice had significantly increased levels of monocyte chemoattractant protein-1. Exposure to gas significantly increased the amount of thymus- and activation-regulated chemokine and macrophage inflammatory proteins-1α in the CLN cells from SBP-immunized mice. These results suggest that Gas components as well as DEP may differentially regulate production of chemokines at local sites.

Intracerebral injection of myelin basic protein (MBP) induces inflammation in brain and causes paraplegia in MBP-sensitized B6 mice.

Brain inflammation and paraplegia can be induced by an additional intraperitoneal (i.p.) and intracerebral (i.c.) restimulation in B6 mice after standard immunization with MBP in Freund's complete adjuvant (FCA) and Bordetella pertussis coadjuvant. Only the combination of i.p. MBP/FCA and i.c. MBP injection could induce clinical paraplegia; either one alone was not effective. Clinical symptoms would develop 2 days after the i.c. injection. The induction of paraplegia was MBP-specific, as irrelevant bovine serum albumin with the same protocol could not induce it. The i.p. restimulation was requisite and needed the MBP in FCA, as MBP in PBS was ineffective. Histopathological observation manifested cellular infiltration by leucocytes in perivascular spaces and cerebral cortex. Neutrophils were prominent at 12 h after i.c. injection, then were replaced by mononuclear cells 24 h later. There were dynamic changes in cell number and immunophenotype of VLA-4+ expression in cervical lymph node cells after i.c. injection. The cells derived from cervical lymph nodes had higher MBP-stimulated proliferation than that of distal lymph nodes. This additional i.p. and i.c. stimulation provides a new manipulation to study brain inflammation.

Early events in peripheral regulatory T cell induction via the nasal mucosa.

Nasal application of soluble Ags leads to Ag-specific suppression of systemic immune responses. This tolerance can be transferred to naive mice by CD4(+) regulatory T cells (T(R) cells) from the spleen, but little is known about the induction of mucosal T(R) cells in vivo. To investigate the induction of T(R) cells in the nose-draining cervical lymph node (CLN), CD4(+) T cells from DO11.10 OVA TCR transgenic mice were transferred to BALB/c recipients. Within 48 h after nasal OVA application, CD4(+) DO11.10 T cells in CLN, but not in the peripheral lymph node, had divided. Similarly, nonmucosal (i.m.) OVA application also induced CD4(+) DO11.10 T cells to proliferate in the draining inguinal lymph node (ILN), yet more vigorously and with different kinetics than the CD4(+) DO11.10 T cells in CLN. Functional analysis revealed that only proliferating CD4(+) DO11.10 T cells from CLN, and not ILN, could transfer tolerance to naive recipients. CD4(+) DO11.10 T cells from CLN were phenotypically similar to CD4(+) DO11.10 T cells from ILN, however, in CLN a higher percentage of CD25(+) proliferating CD4(+) DO11.10 T cells were detected compared with ILN. CD25 is not a discriminative marker for mucosal T(R) cells because both CD25(+) and CD25(-) CD4(+) DO11.10 T cells from the CLN could suppress delayed type hypersensitivity responses in adoptive transfer. These findings demonstrate that although striking similarities exist between the differentiation of T(R) and effector T cells, this does not include their function. We are the first to demonstrate that functional T(R) cells, which reside within both CD25(+) and CD25(-) subsets, can be isolated from CLN as early as 3 days after nasal OVA application.

CC chemokine ligand 3 (CCL3) regulates CD8(+)-T-cell effector function and migration following viral infection.

Chemokines induce the directional migration of targeted populations of leukocytes during periods of inflammation. Moreover, these molecules also regulate T-cell activation and differentiation following antigenic stimulation. In the present study, the contributions of the CC chemokine ligand 3 (CCL3) to the differentiation and migration of effector T cells in response to viral infection of the central nervous system (CNS) were analyzed. CCL3(-/-) mice infected with mouse hepatitis virus exhibited a significant reduction of virus-specific CD8(+) T cells within the CNS, correlating with delayed viral clearance. Decreased infiltration of CD8(+) T cells into infected CCL3(-/-) mice was associated with enhanced accumulation of primed CD8(+) T cells in cervical lymph nodes. Although virus-specific CD8(+) T cells from CCL3(-/-) mice were CD44(high), they remained CD62L(high) and CD25(low), retained CCR7 expression, and contained limited transcripts of the proinflammatory chemokine receptors CCR5 and CXCR3 compared with virus-specific CD8(+) T cells from CCL3(+/+) mice. Furthermore, the absence of CCL3 impaired the cytokine production and cytolytic activity of CD8(+) T cells. In addition, macrophage accumulation within the CNS was significantly decreased in infected CCL3(-/-) mice, correlating with reduced demyelination. These results suggest that CCL3 not only mediates macrophage chemotaxis but also significantly enhances differentiation of primed CD8(+) T cells into effector cells and their release into circulation, thus potentiating effective migration to the site of infection.

Precursors of Borna disease virus-specific T cells in secondary lymphatic tissue of experimentally infected rats.

Borna disease in rats represents an experimental model to study the immunopathological role of T cells in central nervous system disease. Adoptive transfer experiments were performed to investigate homing properties of T cells that infiltrate the brains of infected animals. Lymphocytes isolated from the brains of diseased rats were labelled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and transferred into immunosuppressed infected recipients. In recipient rats displaying neurological disease, labeled lymphocytes were demonstrated in the vicinity of brain cell lesions, suggesting that the neuronal destruction was dependent on the presence of transferred lymphocytes. Furthermore, the presence of virus-specific cytotoxic T cells was scrutinized in secondary lymphatic tissue and the functional activity of lymphocytes isolated from spleens, cervical lymph nodes, and mesenteric lymph nodes of infected animals was tested immediately after isolation and after in vitro restimulation. The data presented here indicate that precursors of Borna disease virus (BDV)-specific CD8(+) T cells are present and cytotoxic activity was demonstrated after in vitro cocultivation with infected cells in cervical lymph nodes and spleens but not in mesenteric lymphoid tissue. Adoptive transfer of in vitro restimulated T cells induced alterations in BDV-infected, immunosuppressed rats that resemble the well-defined clinical symptoms and neuropathology of Borna disease. This report provides for the first time formal evidence that virus-specific cytotoxic T cells are primed in the periphery after BDV infection, a disease that exclusively manifests itself in the central nervous system.

Serum antibody response and nasal lymphoid tissue (NALT) structure in the absence of IL-4 or IFN-gamma.

Immunization via the nasal route is effective for inducing not only mucosal immunity but also antibody (Ab) response in serum. Nasal lymphoid tissue (NALT) is important for induction of systemic immunity. It remains controversial which T effector cell response is important for serum Ab response after nasal immunization. We investigated serum Ab responses and NALT structures in interleukin (IL)-4 gene targeted (IL-4(-/-)) and interferon (IFN)-gamma gene targeted (IFN-gamma(-/-)) mice. Mice were immunized via nostrils with ovalbumin (OVA) and cholera toxin as adjuvant and serum Ab titers were measured 1 week after final antigen challenge. OVA-specific IgG titers in sera of IL-4(-/-) mice indicated a Th(1) type response, whereas titers in IFN-gamma(-/-) mice and wild-type mice indicated a Th(2) type response. Enhanced serum Ab responses were observed in IL-4(-/-) mice but not IFN-gamma(-/-) mice. OVA-specific Ab-forming cells were detected in the cervical draining lymph nodes but were rare or absent in and around the NALT of all strains of mice. Numbers of OVA-specific Ab-forming cells in cervical lymph nodes were significantly higher in IL-4(-/-) mice than in wild-type and IFN-gamma(-/-) mice. Germinal centers of lymphoid follicles were present in NALT of IL-4(-/-) and other mice. Immunohistochemistry for B and T cell markers revealed that NALT of all mice had approximately the same cellular compositions. Although the absence of IL-4 had no effect on NALT structure, IL-4 may suppress induction of serum Ab responses by nasal immunization.

Salmonella enterica serovar Typhi live vector vaccines delivered intranasally elicit regional and systemic specific CD8+ major histocompatibility class I-restricted cytotoxic T lymphocytes.

We investigated the ability of live attenuated Salmonella enterica serovar Typhi strains delivered to mice intranasally to induce specific cytotoxic T-lymphocyte (CTL) responses at regional and systemic levels. Mice immunized with two doses (28 days apart) of Salmonella serovar Typhi strain Ty21a, the licensed oral typhoid vaccine, and genetically attenuated mutants CVD 908 (DeltaaroC DeltaaroD), CVD 915 (DeltaguaBA), and CVD 908-htrA (DeltaaroC DeltaaroD DeltahtrA) induced CTL specific for Salmonella serovar Typhi-infected cells in spleens and cervical lymph nodes. CTL were detected in effector T cells that had been expanded in vitro for 7 days in the presence of Salmonella-infected syngeneic splenocytes. A second round of stimulation further enhanced the levels of specific cytotoxicity. CTL activity was observed in sorted alphabeta+ CD8+ T cells, which were remarkably increased after expansion, but not in CD4+ T cells. CTL from both cervical lymph nodes and spleens failed to recognize Salmonella-infected major histocompatibility complex (MHC)-mismatched cells, indicating that the responses were MHC restricted. Studies in which MHC blocking antibodies were used showed that H-2L(d) was the restriction element. This is the first demonstration that Salmonella serovar Typhi vaccines delivered intranasally elicit CD8+ MHC class I-restricted CTL. The results further support the usefulness of the murine intranasal model for evaluating the immunogenicity of typhoid vaccine candidates at the preclinical level.

IgG plasma cells display a unique spectrum of leukocyte adhesion and homing molecules

AbstractLong-lived antibody-secreting plasma cells are formed in the secondary lymphoid organs and subsequently home to the bone marrow, although the mechanisms that control this migration remain primarily unknown. In this study, we show that IgG plasma cells constitute a significant fraction of cervical lymph node cells from older mice deficient in both E- and P-selectin (E/P−/−), and that these cells can be prospectively isolated by phenotype. These IgG plasma cells were polyclonal, cytoplasmic Ig+, spontaneously secreted antibody, were in the G0/G1 phase of the cell cycle, and failed to express multiple B-cell surface markers. The plasma cells exhibited up-regulated cell surface expression of multiple adhesion molecules, including α4 and leukocyte function-associated antigen 1 (LFA-1) integrins, CD44, and P-selectin glycoprotein ligand 1 (PSGL-1). IgG plasma cells bound to vascular cell adhesion molecule 1 (VCAM-1) significantly better than IgM+B cells, indicating that the α4 integrins were constitutively active. A subset of IgG plasma cells also bound hyaluronic acid, the ligand for CD44. In addition, the IgG plasma cells interacted strongly with E-selectin, but poorly with P-selectin, despite elevated levels of PSGL-1 protein. The preferential interaction of plasma cells with E-selectin, but not P-selectin, correlated with elevated α1,3-fucosyltransferase-VII messenger RNA levels, but selective down-regulation of core 2 β1-6-N-glucosaminyltransferase levels, compared to B cells. These results demonstrate a unique adhesion profile for murine IgG plasma cells. Furthermore, the E/P−/− mice represent a novel system to isolate and purify significant numbers of primary IgG plasma cells.

Detection of disseminated medullary thyroid carcinoma cells in cervical lymph nodes by cytokeratin 20 reverse transcription-polymerase chain reaction.

Local recurrence in differentiated and medullary thyroid carcinoma develops frequently from metastatic infiltration of cervical lymph nodes. Despite an aggressive surgical approach, postoperative calcitonin levels as biochemical evidence for residual cancer cells remain often elevated in patients with medullary thyroid carcinoma. In the present study, we compared the detection rates of disseminated medullary thyroid carcinoma cells in cervical lymph nodes by histopathology with reverse transcription-polymerase chain reaction (RT-PCR) amplification of cytokeratin 20 (CK20) transcripts as a more sensitive but still specific molecular parameter for residual thyroid cancer cells. Forty-two cervical lymph nodes obtained from 7 patients with CK20positive medullary thyroid carcinomas were cut into two halves, one used for conventional histology, the other subjected to RNA extraction and subsequent amplification of cytokeratin 20 transcripts. Matching results for CK20 RT-PCR and histopathology were found in 74% (31/42)of the examined lymph nodes (52% positive results, 48% negative results). Positive CK20 RT-PCR pointed to residual thyroid carcinoma cells in another 19% (8/42), in which no thyroid carcinoma cells were identified by histopathology. Histology and immunohistochemistry,however, identified tumor cells in 7% (3/42) of the analyzed lymph nodes, from which no CK20 transcript could be amplified (false-negative results). These data suggest that CK20 RT-PCR might be more sensitive to detect nodal involvement of CK20 positive medullary thyroid carcinomas than conventional histopathology. In combination with histology, it might help to identify patients with residual disease after surgery.

Nitric oxide‐enhanced resistance to oral candidiasis

A murine model of oral candidiasis was used to show that nitric oxide (NO) is involved in host resistance to infection with Candida albicans in infection-'resistant' BALB/c and infection-'prone' DBA/2 mice. Following infection, increased NO production was detected in saliva. Postinfection samples of saliva inhibited the growth of yeast in vitro. Treatment with NG-monomethyl-L-arginine (MMLA), an inhibitor of NO synthesis, led to reduced NO production, which correlated with an increase in C. albicans growth. Reduction in NO production following MMLA treatment correlated with an abrogation of interleukin-4 (IL-4), but not interferon-gamma (IFN-gamma), mRNA gene expression in regional lymph node cells. Down-regulation of IL-4 production was accompanied with an increase in IFN-gamma production in infection-'prone' DBA/2 mice. There was a functional relationship between IL-4 and NO production in that mice treated with anti-IL-4 monoclonal antibody showed a marked inhibition of NO production in saliva and in culture of cervical lymph node cells stimulated with C. albicans antigen. The results support previous conclusions that IL-4 is associated with resistance to oral candidiasis and suggest that NO is involved in controlling colonization of the oral mucosal surface with C. albicans.

Rotavirus 2/6 virus-like particles administered intranasally in mice, with or without the mucosal adjuvants cholera toxin and Escherichia coli heat-labile toxin, induce a Th1/Th2-like immune response.

We investigated the rotavirus-specific lymphocyte responses induced by intranasal immunization of adult BALB/c mice with rotavirus 2/6 virus-like particles (2/6-VLPs) of the bovine RF strain, by assessing the profile of cytokines produced after in vitro restimulation and serum and fecal antibody responses. The cytokines produced by splenic cells were first evaluated. Intranasal immunization with 50 microg of 2/6-VLPs induced a high serum antibody response, including immunoglobulin G1 (IgG1) and IgG2a, a weak fecal antibody response, and a mixed Th1/Th2-like profile of cytokines characterized by gamma interferon and interleukin 10 (IL-10) production and very low levels of IL-2, IL-4, and IL-5. Intranasal immunization with 10 microg of 2/6-VLPs coadministered with the mucosal adjuvants cholera toxin and Escherichia coli heat-labile toxin (LT) considerably enhanced the Th1/Th2-like response; notably, significant levels of IL-2, IL-4, and IL-5 were observed. Since rotavirus is an enteric pathogen, we next investigated the production of IL-2 and IL-5, as being representative of Th1 and Th2 responses, by Peyer's patch and mesenteric lymph node cells from mice immunized intranasally with 2/6-VLPs and LT. The results were compared to those obtained from splenic and cervical lymph node cells. We found that both cytokines were produced by cells from each of these lymphoid tissues. These results confirm the Th1/Th2-like response observed at the systemic level and show, on the assumption that T cells are the primary cells producing the cytokines after in vitro restimulation, that rotavirus-specific T lymphocytes are present in the intestine after intranasal immunization with 2/6-VLPs and LT.

Minimal residual disease in thyroid carcinoma.

The detection of disseminated tumor cells in differentiated (DTC) and medullary thyroid carcinomas (MTC) is one of the main topics in current thyroid cancer research. Immunocytochemistry and polymerase chain reaction (PCR) provide the tools for the identification of a small number of thyroid cancer cells in peripheral blood and cervical lymph nodes. Thyroid-specific markers, such as thyroglobulin (Tg) mRNA and thyroid peroxidase (TPO) mRNA, have been detected with RT-PCR in blood samples of tumor patients and healthy control subjects. To prevent false-positive results, quantitative PCR systems were established. Tumor-specific markers, such as telomerase activity and cytokeratin 20 (CK20), have been detected in various epithelial tumors. Amplification products of these markers were found in blood samples and in fine-needle aspiration (FNA) biopsies of patients with thyroid carcinomas. Using molecular detection of disseminated tumor cells in cervical lymph nodes with CK20 RT-PCR, a higher percentage of involved lymph nodes was detected compared to immunohistochemistry. The results of the presented studies may help researchers to develop more sensitive methods for early tumor cell dissemination, and refine risk groups that might benefit from more extensive surgical procedures or adjuvant therapy. However, the prognostic value of minimal residual disease (MRD) in thyroid carcinoma has to be confirmed in large or multicenter prospective studies.

A Serine Protease Inhibitor, N-α-Tosyl-[formula omitted]-lysine Chloromethyl Ketone, Prolongs Rat Hepatic Allograft Survival

Background. Serine protease inhibitors have profound suppressive effects on cellular and humoral immune responses. We investigated the effect of a serine protease inhibitor, N-α-tosyl-l-lysine chloromethyl ketone (TLCK), on hepatic allograft survival in rats.Methods. Orthotopic hepatic transplantation was performed in an ACI (RT1a)-to-LEW (RT11) rat combination. TLCK was administered continuously at a dose of 4.4 mg/kg/day using an osmotic subcutaneous infusion minipump.Results. TLCK prolonged hepatic allograft survival. Histologic staging of acute rejection based on Banff criteria in TLCK-treated hepatic allografts was significantly lower than in untreated allografts. TLCK significantly reduced serum concentrations of interferon (IFN)-γ and tumor necrosis factor (TNF) α in allograft recipients. TNF-α mRNA levels in TLCK-treated allografts were significantly lower than in untreated allografts. TLCK also decreased perforin mRNA levels in hepatic allografts. Hepatic infiltrates eluted from TLCK-treated allografts showed significantly lower cell-mediated lympholytic activity against donor Con A blast cervical lymph node cells than those from untreated allografts. In vitro, TLCK suppressed interleukin-2 production and [3H]thymidine incorporation into an allogeneic mixed lymphocyte reaction.Conclusion. TLCK suppressed acute allograft rejection, suggesting a novel immunosuppressive strategy for therapy of acute organ rejection.