Matrix Metalloproteinase and Cytokine Profiles in Monocytes over the Course of Stroke

Stroke is a common cause of death and disability in our society. Stroke is associated with changes in immune responses within the central nervous system as well as systemically. The cells contributing to such changes as well as the factors contributing to formation of the inflammatory infiltrate observed in stroke remain to be clarified. In this study, blood monocytes and corresponding mononuclear cells (MNC) were separated and examined in parallel within 4 days and 1–3 months after onset of ischemic stroke. Numbers of TNF--, IL-12-, IL-6-, and IL-10-secreting cells and of cells expressing mRNA for matrix metalloproteinase (MMP)-1, -2, -7, -9 and tissue inhibitor of MMP (TIMP)-1 were studied. The TNF--, IL-12-, and IL-6-secreting monocytes and MNC were elevated during the acute phase compared to healthy controls. Such differences were not observed when stroke patients were examined during convalescence. The IL-10-secreting monocytes did not change over the course of stroke. Levels of monocytes expressing MMP-1, MMP-7 and TIMP-1 mRNA were elevated in the acute phase of stroke patients compared to convalescence and healthy controls, as were levels of MMP-1, -2, -7, -9 and TIMP-1 mRNA expressing blood MNC. The MMP-2 and -9 activity as measured by zymography also was higher in MNC supernatants in the acute phase of stroke compared to convalescence. The high levels of proinflammatory cytokines and MMPs in blood monocytes and MNC further demonstrate the presence of systemic aberrations in the acute phase of stroke. Such changes may contribute to the influx of blood-borne cells into the ischemic lesions during the acute phase of stroke.     

Human T cell responses to HPV 16 E2 generated with monocyte-derived dendritic cells.

Persistent infection with human papillomavirus (HPV) type 16 has been implicated in the etiology of cervical cancer. The E2 protein is required early in viral infection and therefore may serve as a useful immune target for a vaccine aimed at prevention or therapy of premalignant lesions. Dendritic cells (DC) prepared from monocytes and pulsed with bacterially produced HPV 16 E2 C-terminus protein were used to stimulate autologous T cells over several rounds of stimulation. T cells were tested for gamma-interferon release by ELISPOT and for cytotoxic activity by (51)chromium release assays. To generate E2-expressing target cells for cytotoxicity assays, we constructed a recombinant vaccinia virus encoding HPV 16 E2, which was used to infect autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL). The results show that DC pulsed with E2 C-terminus protein induce gamma-interferon-releasing T cells as demonstrated by ELISPOT. Furthermore, we demonstrate E2-specific lysis of vaccinia-E2 infected autologous LCL by CD8+ cytotoxic T lymphocytes (CTL). E2-specific CTL did not lyse untreated autologous LCL or LCL infected with wild-type vaccinia and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. In addition, T cells stimulated with DC in the absence of E2 failed to demonstrate lysis of vaccinia-E2-labeled targets. Phenotypically, CTL populations were CD3+/CD8+. These results will facilitate the study of naturally occurring T-cell responses to HPV E2 in patients with cervical intraepithelial neoplasia and the development of immunotherapeutic strategies designed to treat this and other HPV-associated diseases.     

25‐OH‐Vitamin D Deficiency and Cellular Alloimmunity as Measured by Panel of Reactive T Cell Testing in Dialysis Patients

Primed antidonor alloreactive T cells are detrimental to transplant outcome, but factors that impact the strength of this immune response prior to transplantation are unknown. We tested peripheral blood mononuclear cells from dialysis patients, against panels of allogeneic, primary B‐cell lines in a newly standardized IFNγ ELISPOT panel of reactive T cell (PRT) assay. Results were correlated with known alloantibody‐sensitizing events and other clinical parameters. As 25‐OH‐vitamin D deficiency is associated with enhanced cellular immunity, is common in dialysis patients and is correctable, we assessed the relationship between serum 25‐OH‐vitamin D and the PRT. Using independent test and validation cohorts we found that low serum levels of 25‐OH‐vitamin D (<26 ng/mL) correlated with high‐PRT values (in the upper 50th percentile, OR 0.02, p = 0.01) independent of age, sex, race, previous transplant, transfusion, pregnancy, time on dialysis, panel of reactive antibody, iPTH, and treatment with 1,25‐OH‐vitamin D. The data provide a potential mechanism for the possible relationship between vitamin D deficiency and poor posttransplant outcome, and support studies to test the impact of 25‐OH‐vitamin D repletion on alloimmunity and allograft injury in kidney transplant candidates.     

Cytomegalovirus vaccination of leukemia and lymphoma patients after allogeneic stem cell transplantation — Validation of a peptide vaccine

Peptide vaccination constitutes a novel immunotherapeutical approach for the treatment of patients with solid tumors, lymphoma and leukemia. Moreover it might be of use in hematooncological patients for the prevention and therapy of infections like cytomegalovirus (CMV) reactivation due to immunosuppression. To meet good manufacturing practice (GMP) criteria, we introduce here a bio-assay to validate peptide vaccines for peptide content and bio-activity.As a paradigm for peptide vaccine preparation the immunogenic CMV peptide 495-503 NLVPMVATV lyophilisate was resolubilized in dimethyl sulfoxide, phosphate buffered saline and admixed with Montanide™.Addition of different amounts of peptide (10-80 µg) to a mixed lymphocyte peptide culture (MLPC) resulted in the generation of interferon (IFN) gamma and granzyme B releasing CD8⁺ CMV tetramer⁺ T cells in a dose dependent manner. The combination of FACS and ELISPOT results allowed the definition of the peptide amount in a vaccine preparation. Storage at ± 4 °C over 24 h did not result in a significant change of the immunogenicity of the vaccine. In contrast, cryopreservation of the vaccine at − 20 °C resulted in a loss of immunogenicity.Quantitation of tumor/viral antigen peptides admixed with adjuvants, such as incomplete Freund's adjuvant (IFA), is feasible through bio-assays as the modified ELISPOT/FACS assay described here, meeting GMP criteria for multi-center trials.     

MIG (CXCL9) is a more sensitive measure than IFN-gamma of vaccine induced T-cell responses in volunteers receiving investigated malaria vaccines

For many years the IFN-γ ex vivo ELISPOT has been a major assay for assessing human T-cell responses generated by malaria vaccines. The ELISPOT assay is a sensitive assay, but an imperfect correlate of protection against malaria. Monokine induced by gamma (MIG), or CXCL9, is a chemokine induced by IFN-γ and has the potential to provide amplification of the IFN-γ signal. MIG secretion could provide a measure of bio-active IFN-γ and a functional IFN-γ signalling pathway. We report that detecting MIG by flow cytometry and by RT-PCR can be more sensitive than the detection of IFN-γ using these methods. We also find that there is little inter-individual variability in MIG secretion when detected by flow cytometry and that the MIG assay may be used to estimate the amount of bio-active IFN-γ present. Measurement of MIG alongside IFN-γ may provide a fuller picture of Th1 type responses post-vaccination.     

Identification of a major histocompatibility complex class I-restricted T-cell epitope in the tumour-associated antigen, 5T4

5T4 is a surface glycoprotein expressed on placental trophoblasts and also on a wide range of human carcinomas. Its highly restricted expression on normal tissues and broad distribution on many carcinomas make 5T4 a promising target for cancer immunotherapy. In the current study, we set out to investigate whether a 5T4-specific cytotoxic T lymphocyte (CTL) repertoire exists in healthy individuals. CD4-depleted peripheral blood mononuclear cells (PBMCs) from blood donors were screened using an ex vivo interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay. A panel of overlapping peptides, spanning the full length of the 5T4 protein, was used as a source of antigen. In the process of screening, one out of 30 blood donors demonstrated a positive ex vivo IFN-gamma ELISPOT response to a single 5T4 peptide. A polyclonal T-cell line was derived from this donor by culturing PBMCs with autologous peptide-pulsed dendritic cells (DCs). The resulting polyclonal T-cell line and clones were tested in a 51Cr-release assay and by ELISPOT and were shown to be peptide specific. Furthermore, antigen-presenting cells (APCs), infected with a viral vector expressing 5T4, were able to stimulate IFN-gamma production by the peptide-specific T-cell clones. A minimal CD8 epitope, PLADLSPFA, has been identified and found to be restricted through human leucocyte antigen (HLA) Cw7. Subsequently, we have demonstrated that HLA-Cw7-positive colorectal cancer patients vaccinated with a recombinant vaccinia viral vector encoding 5T4 (TroVax) are capable of mounting a strong IFN-gamma ELISPOT response to this novel CTL epitope. These findings have potential application in cancer immunotherapy in terms of subunit vaccine design and the monitoring of immune responses induced in patients by 5T4-based therapies.     

Effect of probiotics and breastfeeding on the bifidobacterium and lactobacillus/enterococcus microbiota and humoral immune responses

OBJECTIVE: To assess impact of probiotics and breastfeeding on gut microecology. STUDY DESIGN: Mothers were randomized to receive placebo or Lactobacillus rhamnosus GG before delivery, with treatment of the infants after delivery. We assessed gut microbiota, humoral immune responses, and measured soluble cluster of differentiation 14 (sCD14) in colostrum in 96 infants. RESULTS: Fecal Bifidobacterium and Lactobacillus/Enterococcus counts were higher in breastfed than formula-fed infants at 6 months; P <.0001 and P=.01, respectively. At 3 months, total number of immunoglobulin (Ig)G-secreting cells in breastfed infants supplemented with probiotics exceeded those in breastfed infants receiving placebo; P=.05, and their number correlated with concentration of sCD14 in colostrum. Total numbers of IgM-, IgA-, and IgG-secreting cells at 12 months were higher in infants breastfed exclusively for at least for 3 months and supplemented with probiotics as compared with breastfed infants receiving placebo; P=.005, P=.03 and P=.04, respectively. Again, sCD14 in colostrum correlated with numbers of IgM and IgA cells; P=.05 in both. CONCLUSIONS: We found an interaction between probiotics and breastfeeding on number of Ig-secreting cells, suggesting that probiotics during breastfeeding may positively influence gut immunity.     

Oral immunization with recombinant Lactococcus lactis NZ9000 expressing human papillomavirus type 16 E7 antigen and evaluation of its immune effects in female C57BL/6 mice

The ORFs of both native and codon-optimized E7 genes were successfully fused to SPusp45 signal peptide and expressed by a nisin-controlled gene expression system in the NZ9000 strains of Lactococcus lactis. Recombinant strains were confirmed by Western blot analysis. To measure immune responses against the E7 antigen, specific-pathogen-free C57BL/6 mice were inoculated with L lactis harboring pNZ8123-rE7 by oral gavage. Then, specific antibodies and cytokines were measured by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay, respectively. Oral administration of L lactis strains expressing rE7 elicited the highest levels of E7-specific antibody and greatest numbers of E7-specific CD4⁺ T helper and CD8⁺ T cell precursors. Our outcomes indicated that the HPV-16 E7 specific IL-2- and IFN-γ-secreting T cells in antigen-stimulated splenocytes and intestinal mucosal lymphocytes were significantly higher than the control groups. Our data also demonstrated that mice vaccinated with recombinant L lactis were able to generate potent protective effects against challenge with the E7-expressing tumor cell line (TC-1). Moreover, L lactis containing pNZ8123-HPV16-optiE7 showed strong therapeutic antitumor effects against established tumors in vivo. These findings demonstrate that recombinant L lactis induce both humoral and cellular immune responses in mice and are therefore recommended for therapeutic treatments in humans after oral administration.     

涂阳活动性肺结核患者HRCT评分与细菌学及免疫学相关性分析

目的探讨HRCT评分标准在诊断活动性肺结核中的应用价值。方法对42例痰涂阳性肺结核患者,进行HRCT检查和评分,每侧肺组织划分为为三个区域,分别对各类型CT征象所累及范围进行评分;分析评分结果与痰细菌学检查结果和外周血结核菌特异性IFN—y水平之间的相关性。结果根据结核菌涂片结果分为4组,其中,AFB（＋）12例、AFB（＋＋）11例、AFB（＋＋＋）10例、AFB（＋＋＋＋）9例,相关性分析显示（1）HRCT总评分与痰阳性分级有正相关性（r=0．9661 P〈0．0001）：（2）HRCT总评分与结核菌抗原ESAT6、P4-6、P8．10特异性IFN—y释放水平均呈正相关（r=0．4805,P=0．0017,r=0．4451,P=0．0083,r=0．4211,P=0．0131）。结论以活动性肺结核患者复杂的CT征象的范围为基础的HRCT评分标准与细菌学及免疫学均呈正相关,表明该HRCT评分标准对临床诊断治疗和疗效判定具有一定指导意义。