Neisseria lactamica attenuates TLR-1/2-induced cytokine responses in nasopharyngeal epithelial cells using PPAR-γ.

The upper respiratory tract commensal Neisseria lactamica (Nlac) induces protective humoral immunity against pathogenic Nmen serogroup B (Nmen), but whether it also affords anti-inflammatory mucosal protection, as reported for several gut commensals, has not been investigated. Here we demonstrate for the first time that Nlac weakly induces inflammatory responses compared with Nmen in the nasopharyngeal epithelial cell line, Detroit 562, and that Nlac achieves this by attenuation of secretory cytokine (TNF-α and IL-6) and to a lesser extent chemokine (IL-8 and RANTES) responses. Culture of Detroit cells with Nlac inhibited the induction of cytokine-chemokine mRNA by Nmen, reduced Nmen-induced NF-κß activity and increased constitutive PPAR-γ protein expression. Pretreatment of Detroit cells with a PPAR-γ antagonist abrogated the attenuation of inflammatory IL-6 by Nlac, as did heat-killing of the organisms and preventing their invasion with cytochalasin D. Inflammatory responses from Detroit cells were readily attenuated by Nlac following stimulation with pathogenic Nmen but more specifically following stimulation with the TLR-1/2 agonist Pam3Cys and pro-inflammatory cytokines (IL-1ß, TNF-α) but not LTA or LPS. These results indicate that Nlac plays an important role in suppressing pathogen-induced inflammation in the nasopharyngeal mucosa, mediated through TLR-1/2 stimulation, by activating PPAR-γ and inhibiting NF-κß activity.

A phase I clinical, pharmacological, and biological trial of interleukin 6 plus granulocyte-colony stimulating factor after ifosfamide, carboplatin, and etoposide in children with recurrent/refractory solid tumors: enhanced hematological responses but a high incidence of grade III/IV constitutional toxicities.

A Phase I trial was conducted to determine the safety, biological activity, and hematopoietic recovery by the combination of interleukin 6 (IL-6) and granulocyte-colony stimulating factor (G-CSF) after myelosuppressive chemotherapy in children. Patients <22 years of age at diagnosis with either recurrent or refractory solid tumors received ifosfamide 1,800 mg/m2/day x 5 days, carboplatin 400 mg/m2/ day x 2 days, and etoposide 100 mg/m2/day x 5 days, followed by daily s.c. G-CSF (5 microg/kg/day) and IL-6 (2.5, 3.75, or 5.0 microg/kg/day). Pharmacokinetic, proinflammatory mediator levels, hematopoietic colony assays, and cytokine receptor expression studies were performed during course one. Nineteen patients were evaluable for toxicity and received IL-6 at doses of 2.5 (n = 8), 3.75 (n = 5), or 5.0 (n = 6) microg/kg/day. Dose-limiting constitutional toxicity occurred in two of six patients at 5.0 microg/kg/day, two of five patients at 3.75 microg/kg/day, and two of eight patients at 2.5 microg/kg/day. The maximum tolerated dose (MTD) exceeded the lowest dose tested. Because of lack of drug availability, an MTD was not established. The maximum concentration of IL-6 (2.5 microg/kg/day) was 0.799 +/- 1.055 ng/ml (mean +/- SD). During the first course, the median time to absolute neutrophil count > or = 1,000/mm3 and platelets > or = 100,000 mm3 was estimated at 19 and 23 days, respectively. Peripheral blood progenitor cells expressing receptors to IL-3, IL-6, and G-CSF increased significantly over baseline (P < 0.05). After the first dose of IL-6, IFN-gamma levels were abnormal in 13 patients, and IL-1beta levels were abnormal in 10 patients. IL-6 has a high incidence of constitutional toxicity and a lower MTD in children compared with adults. In vivo use of IL-6 in children after chemotherapy remains limited. However, IL-6 may be more optimally investigated in children under ex vivo conditions.

Immunologic protection afforded by sunscreens in vitro.

Several studies have suggested a lack of correlation between sunscreen sun protection factor and protection of the skin immune system, potentially allowing greater damage to the skin by removing the natural protective erythemal response to sun exposure. Despite this, routine testing of immune protection afforded by sunscreens is not performed by industry. Current laboratory methods for investigating the efficacy of sunscreen protection of epidermal immune function use the induction of contact hypersensitivity or epidermal cell alloantigen presentation. Animal models, cell culture systems, and in vivo human studies are commonly employed, but all these systems have significant drawbacks for use in routine testing. The purpose of this study was to develop an in vitro system for testing the immunologic protection afforded by sunscreens in human skin. Five test sunscreens plus a vehicle control were tested in a "blind" fashion for their in vitro level of immune protection. Creams were applied in a standard manner to human whole skin explants and were irradiated over a range of physiologic doses using an Oriel solar simulator. A mixed epidermal lymphocyte reaction was used to quantify epidermal alloantigen-presenting capacity, in the presence or absence of test cream, for five explants. Results consistently demonstrated that all the test sunscreens protected beyond their designated sun protection factors, whereas the vehicle conferred no protection. The explant-mixed epidermal lymphocyte reaction system gave consistent, reproducible results and may prove useful for the allocation of an immune protection factor to all sunscreens.

Autologous bone marrow transplantation for childhood acute lymphoblastic leukemia: a novel combined approach consisting of ex vivo marrow purging, modulation of multi-drug resistance, induction of autograft vs leukemia effect, and post-transplant immuno- and chemotherapy (PTIC).

In an attempt to reduce the high relapse rate associated with ABMT, five children with high-risk first CR and 19 in second or subsequent CR lacking matched family allogeneic donors underwent ABMT with chemopurged bone marrow utilizing verapamil (VPL), vincristine, and VP-16. Patients were conditioned with TBI, VPL bolus and infusion with VP-16 and cyclophosphamide. The first cohort of patients (n = 4) received only cyclosporin A (CsA). The second cohort (n = 7) received CsA and alpha interferon (total = 11 with post-transplant immunotherapy alone.) The third cohort (n = 13) received CsA and six alternating cycles of alphaIFN and chemotherapy and six additional cycles of chemotherapy (vincristine, VP-16, Ara-C, prednisone) followed by G-CSF (post-transplant immune chemotherapy (PTIC)). The 2-year DFS is 42+/-10% (90% confidence interval (CI) is 26.5-58.5%) and 2-year overall survival is 54+/-10% (90% CI is 37.5-70.5%). Furthermore, patients receiving PTIC (n = 13) vs immunotherapy alone (CsA+/-aIFN) (n = 11) had a substantially better 2 year DFS and OS: 69+/-13% vs 13+/-12% and 85+/-10% vs 25+/-15% (P = 0.008 and P = 0.06, respectively). These results suggest that the use of ABMT with chemopurging, combined with PTIC is well tolerated and may be an alternative new approach in the treatment of a subset of children with high-risk first CR or > or = second CR ALL who lack closely matched family-related allogeneic donors.

p53 induction in normal human skin in vitro following exposure to solar simulated UV and UV-B irradiation.

Exposure of normal human breast skin ex vivo to physiological levels of UV-B and solar simulated UV results in a UV dose- and time-dependent increase in epidermal p53, as determined by PAGE analysis. Peak p53 levels are detected 12 to 24 h post irradiation with UV-B (470-1410 mJ cm-2) and solar simulated UV (5-12 minimal erythema dose (MED) equivalents). Irradiation with an FS20 UV-B lamp, contaminated with UV-A and UV-C (74-1111 mJ cm-2), also induces peak levels after 12 h incubation at 37 degrees C but these levels persist to 36 h post UV irradiation. In all cases p53 levels start to return to normal by 48 h culture. A significant positive correlation is demonstrated between UV-B dose (47-1645 mJ cm-2) and p53 level (p < 0.01, R > 0.977) in explants cultured for 24 h at 37 degrees C post irradiation. The FS20 induces a 'UV-B' dose-dependent increase in p53 to a maximum from 370 to 1111 mJ cm-2. Similarly, solar simulated UV induces a plateau of peak p53 induction between 5 and 15 MED equivalents. Immunohistochemical analysis using microwave retrieval on 5 microns sections shows the same pattern of p53 staining with UV-B and solar UV insult, but proves unreliable as a method of quantification. These results suggest that the skin explant model may be a useful tool in the evaluation of UV-induced epidermal cell damage, providing a valuable alternative to in vivo studies.

Influence of age and carriage status on salivary IgA to Neisseria meningitidis.

Asymptomatic carriage of Neisseria meningitidis is common (5-35% of individuals) while the incidence of invasive meningococcal disease is fairly low (<1-5 per 100,000 per annum in Europe). Naturally acquired protective immunity may account for this difference. In this study, we investigated the relationship between anti-meningococcal salivary IgA and age and carriage. We showed that salivary IgA to a range of meningococcal antigens increased successively with age with some specificity for commonly circulating serosubtypes. In a group of 258 students 37 (14%) of whom were carriers of N. meningitidis serogroup B, higher levels of specific IgA were associated with carriage. Stratified analysis revealed a positive relationship between smoking and specific anti- N. meningitidis IgA independent of current carriage, weighted odds ratio (OR) 4.1 (95% CI 1.1-18) and OR 3.8 (95% CI 0.96-16) for reference strains B:1:P1.14 and B:4:P1.5,4 respectively. These data implicate IgA as a factor in host defence from meningococcal invasion, although the precise mechanisms remain uncertain.

Determination of molecular weights by differential cryoscopy on small volumes of dilute solutions of oligopeptides.

A differential cryoscope of the equilibrium type is described. It requires only 1 ml of sample for a molecular-weight determination of a solute in a solvent that freezes below room temperature. The instrument is sensitive to +/- 0.0002 degrees C, which corresponds to +/- 0.0001 mol of solute per 1000 g of water. The apparatus was evaluated by measuring the freezing point depressions of 0.015 M urea and 0.01 M alanine in water, the measured molecular weights being accurate to within +/- 5%. The molecular weights of the following oligopeptides were then measured to determine their states of aggregation in the cited solvents: Ac-Asn-Pro-Tyr-NHMe in H2O and dimethyl sulfoxide and cyclo(L-alanyl-L-alanyl-epsilon-aminocaproyl), cyclo(L-alanyl-D-alanyl-epsilon-aminocaproyl), cyclo(L-alanyl-L-analyl-omega-capryl), cyclo(L-alanyl-D-alanyl-omega-capryl), and Ac-Tyr-Pro-Asn-NHMe in dimethyl sulfoxide.

Successful in vivo immunolocalization of Langerhans cell histiocytosis with use of a monoclonal antibody, NA1/34.

The antibody NA1/34 is a murine monoclonal antibody directed against the CD1a surface antigen expressed on normal Langerhans cells, cortical thymocytes, and on lesional cells in Langerhans cell histiocytosis (LCH). Our hypothesis was that NA1/34 would localize sites of disease activity in patients with multisystem LCH. To test this hypothesis, indium 111-labeled NA1/34 was administered to five patients with multisystem LCH and serial gamma scans were obtained for up to 120 hours. Serial serum samples were obtained from one patient for analysis of anti-mouse Ig antibody and NA1/34 levels. Direct and indirect immunofluorescence staining for CD1a and NA1/34 were performed on a tissue biopsy specimen from one patient after administration of the antibody. The 1- and 4-hour scans showed distribution of antibody in the blood pool, but in later scans localization of the antibody was noted in areas of known disease activity in all five patients. Bony lesions, previously seen on skeletal radiographs, were especially well identified. Serum kinetics studies showed clearance of the antibody from the blood pool within 12 hours of administration. Direct binding of NA1/34 to lesional cells was demonstrated by direct immunofluorescence. The only adverse effect was urticaria in one patient. We conclude that NA1/34 localizes disease activity in vivo in bones of patients with LCH with minimal toxic effects. An evaluation of its role in determining disease extent ("staging") and in treatment is now needed.

A comparison of ex vivo expanded DCs derived from cord blood and mobilized adult peripheral blood plastic-adherent mononuclear cells: decreased alloreactivity of cord blood DCs.

BACKGROUND: Cord blood (CB) has been used as an alternative source of transplantable allogeneic stem cells for a variety of malignant and non-malignant diseases. However, we have demonstrated delayed recovery of T- and B-cell function, and T-cell subsets post unrelated CB transplantation (UCBT), and deficiencies of CB mononuclear cells (MNC) in producing cytokines, including G-CSF, GM-CSF, M-CSF, IL-12, and IL-15. In this study we have investigated the ex vivo generation of DC from CB versus mobilized adult peripheral blood (APB) for later use as adoptive cellular immunotherapy. METHODS: CB and APB-adherent MNC were cultured in serum-free media with GM-CSF IL-4, FLT-3 ligand, tumor growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha) for 7 days. Morphology, phenotype, immunohistochemistry, clonogenic activity, and alloreactivity in MLR were evaluated. RESULTS: CB and APB monocyte-derived ex vivo expanded DC expressed similar DC markers CD83 (31.27+ 11.7% versus 34.0+ 5.2%, CB versus APB), CD1a (23.4+ 4.2% versus 27.6+ 6.3%), and CD80 (21.97+ 12.01% versus 27.7+ 5.95). Immunohistochemistry showed that cells with DC morphology expressed CDla but not CD14. Neither FLT-3 ligand nor TGF-fl enhanced DC expansion. Addition of 10% autologous plasma to CB cultures promoted greater cell survival and a 150% increase in CDla + /CD80+ cell recovery. CB DC were 62% as effective stimulators of adult allogeneic T-cels as APB DC (p < .05) in allogeneic MLR. DISCUSSION: While phenotypically similar, CB and APB DC have differential potency in allogeneic MLR, which may account for the difference in GvHD and infection incidence and severity between UCBT and allogeneic stem cell transplantation, and may require a different approach for adoptive cellular immunotherapy. The mechanism(s) associated with these differences require further elucidation.

Feasibility study of IL-11 and granulocyte colony-stimulating factor after myelosuppressive chemotherapy to mobilize peripheral blood stem cells from heavily pretreated patients.

PURPOSE: Pediatric patients with solid tumors treated with prolonged dose-intensive chemoradiotherapy are poor mobilizers of peripheral blood stem cells (PBSC). We have conducted a pilot study to mobilize PBSC in eight pediatric patients with relapsed solid tumors using ifosfamide, carboplatin, and etoposide (ICE) followed-up by IL-11 plus granulocyte colony-stimulating factor (G-CSF). PATIENTS AND METHODS: Patients received ifosfamide 1.8 g/m2 per day for 5 days, carboplatin 400 mg/m2 per day for 2 days, and etoposide 100 mg/m2 per day for 5 days. After completion of ICE chemotherapy, patients received daily subcutaneous injections of G-CSF (5 microg/kg per day) and IL-11 (50-100 microg/kg per day) until peripheral stem cell apheresis. RESULTS: The median age was 11 years. Diagnosis included three relapsed Hodgkin disease, three relapsed central nervous system tumors, one relapsed Wilms tumor, and one relapsed rhabdomyosarcoma. The median number of apheresis procedures required to obtain 5 x 10(6) CD34+ cells/kg was one. The mean +/- standard error of mean (SEM) total CD34+ cells collected was 14.0+/-2.7 x 10(6)/kg. The mean +/- SEM total CD34+/CD41+ cells collected was 4.6+/-1.9 x 10(6)/kg. Seven of the eight patients have subsequently undergone myeloablative chemotherapy with autologous PBSC transplantation and have reconstituted hematopoiesis with a median time to neutrophil recovery of 10 days and platelet recovery of 15.5 days. CONCLUSIONS: We conclude that the regimen of ICE/IL-11 plus G-CSF is successful in mobilizing large numbers of CD34+ PBSC cells with a limited number (one) of apheresis collections in patients that have previously been heavily pretreated with chemotherapy/radiotherapy.

Ifosfamide, carboplatin and etoposide in children with poor-risk relapsed Wilms' tumor: a Children's Cancer Group report.

BACKGROUND: The outcome of children with relapsed Wilms' tumor is poor, especially with poor-risk factors such as unfavorable histology, early recurrence, previous three-drug therapy, relapse not confined to lungs and abdominal relapse following abdominal radiotherapy. We report the overall response rate, progression-free survival and overall survival of 11 children with relapsed and poor-risk Wilms' tumor following ifosfamide/carboplatin/etoposide (ICE) chemotherapy. PATIENTS AND METHODS: ICE therapy consisted of ifosfamide 1800 mg/m2/day (on day 0-4), carboplatin 400 mg/m2/day (on day 0-1) and etoposide 100 mg/m2/day (on day 0-4). The median age at diagnosis was 39 months (range from 13 months to 16 years) and the median time to relapse after initial diagnosis was 9 months (range 4-72 months). All but one patient had at least one poor prognostic feature, with eight patients showing three or four. RESULTS: After ICE chemotherapy the number of patients showing a complete response (CR) was three (27%) and a partial response (PR) was six (55%). The overall response rate (CR+PR) was 82%. Five of the six patients with a PR subsequently achieved a CR with further therapy. The 3-year event-free survival and overall survival were 63.6 +/- 14.5%. CONCLUSIONS: The response rate in children with relapsed and poor-risk Wilms' tumor is >80% with ICE re-induction chemotherapy followed by post-ICE therapy. The optimal approach for post-ICE consolidation therapy has yet to be determined.