The impact of immune response on endochondral bone regeneration.

Tissue engineered cartilage substitutes, which induce the process of endochondral ossification, represent a regenerative strategy for bone defect healing. Such constructs typically consist of multipotent mesenchymal stromal cells (MSCs) forming a cartilage template in vitro, which can be implanted to stimulate bone formation in vivo. The use of MSCs of allogeneic origin could potentially improve the clinical utility of the tissue engineered cartilage constructs in three ways. First, ready-to-use construct availability can speed up the treatment process. Second, MSCs derived and expanded from a single donor could be applied to treat several patients and thus the costs of the medical interventions would decrease. Finally, it would allow more control over the quality of the MSC chondrogenic differentiation. However, even though the envisaged clinical use of allogeneic cell sources for bone regeneration is advantageous, their immunogenicity poses a significant obstacle to their clinical application. The aim of this review is to increase the awareness of the role played by immune cells during endochondral ossification, and in particular during regenerative strategies when the immune response is altered by the presence of implanted biomaterials and/or cells. More specifically, we focus on how this balance between immune response and bone regeneration is affected by the implantation of a cartilaginous tissue engineered construct of allogeneic origin.

Report of three imported cases of neurocysticercosis in Guadeloupe.

BACKGROUND: Neurocysticercosis is endemic in most countries of Central and South America but has rarely been described in the French West Indies. We aimed to better understand the clinical and radiological presentation of our cases. CASE PRESENTATION: We report three cases of neurocysticercosis in patients living in Guadeloupe, with different clinical and radiological presentations. CONCLUSION: Given the eventuality of autochtonous transmission, the diagnosis should be considered in all patients living in Guadeloupe presenting with seizures.

Implementation of latent tuberculosis screening in HIV care centres: evaluation in a low tuberculosis incidence setting.

The screening and treatment of latent tuberculosis infection (LTBI) to prevent active tuberculosis (TB) is recommended by the WHO in all HIV-infected patients. The aim of this study was to evaluate its implementation within Belgium's HIV care. A multiple-choice questionnaire was sent to 55 physicians working in the country's AIDS reference centres. Response rate reached 62%. Only 20% screened all their HIV-infected patients for LTBI. Screening methods used and their interpretation vary from one physician to another. The main barriers to the implementation of LTBI screening and treatment, as perceived by the participants, are lack of sensitivity of screening tools, risks associated with polypharmacy and toxicity of treatment. The poor coverage of LTBI screening reported here and the inconsistency in methods used raises concern. However, this was not unexpected as, in low-TB incidence countries, who, when and how to screen for LTBI remains unclear and published guidelines show important disparities. Recently, a targeted approach in which only HIV-infected patients at highest risk of TB are screened has been suggested. Such a strategy would limit unnecessary exposure to LTBI treatment. This methodology was approved by 80% of the participants and could therefore achieve greater coverage. Its clinical validation is still pending.

Early cellular immune response to a new candidate mycobacterial vaccine antigen in childhood tuberculosis.

The search for novel vaccines against tuberculosis (TB) would benefit from in-depths knowledge of the human immune responses to Mycobacterium tuberculosis (Mtb) infection. Here, we characterised in a low TB incidence country, the immune responses to a new candidate vaccine antigen against TB, the heparin-binding haemagglutinin (HBHA), in young children in contact with an active TB case (aTB). Children with no history of BCG vaccination were compared to those vaccinated at birth to compare the initial immune responses to HBHA with secondary immune responses. Fifty-eight children with aTB and 76 with latent TB infection (LTBI) were included and they were compared to 90 non-infected children. Whereas Mtb-infected children globally secreted more interferon-gamma (IFN-γ) in response to HBHA compared to the non-infected children, these IFN-γ concentrations were higher in previously BCG-vaccinated compared to non-vaccinated children. The IFN-γ concentrations were similar in LTBI and aTB children, but appeared to differ qualitatively. Whereas the IFN-γ secretion induced by native methylated and recombinant non-methylated HBHA were well correlated for aTB, this was not the case for LTBI children. Thus, Mtb-infected young children develop IFN-γ responses to HBHA that are enhanced by prior BCG vaccination, indicating BCG-induced priming, thereby supporting a prime-boost strategy for HBHA-based vaccines. The qualitative differences between aTB and LTBI in their HBHA-induced IFN-γ responses may perhaps be exploited for diagnostic purposes.

Long-incubation-time gamma interferon release assays in response to purified protein derivative, ESAT-6, and/or CFP-10 for the diagnosis of Mycobacterium tuberculosis infection in children.

The diagnosis of childhood active tuberculosis (aTB) and latent Mycobacterium tuberculosis (M. tuberculosis) infection (LTBI) remains a challenge, and the replacement of tuberculin skin tests (TST) with commercialized gamma interferon (IFN-γ) release assays (IGRA) is not currently recommended. Two hundred sixty-six children between 1 month and 15 years of age, 214 of whom were at risk of recent M. tuberculosis infection and 51 who were included as controls, were prospectively enrolled in our study. According to the results of a clinical evaluation, TST, chest X ray, and microbiological assessment, each children was classified as noninfected, having LTBI, or having aTB. Long-incubation-time purified protein derivative (PPD), ESAT-6, and CFP-10 IGRA were performed and evaluated for their accuracy in correctly classifying the children. Whereas both TST and PPD IGRA were suboptimal for detecting aTB, combining the CFP-10 IGRA with a TST or with a PPD IGRA allowed us to detect all the children with aTB with a specificity of 96% for children who were positive for the CFP-10 IGRA. Moreover, the combination of the CFP-10 IGRA and PPD IGRA detected 96% of children who were eventually classified as having LTBI, but a strong IFN-γ response to CFP-10 (defined as >500 pg/ml) was highly suggestive of aTB, at least among the children who were <3 years old. The use of long-incubation-time CFP-10 IGRA and PPD IGRA should help clinicians to quickly identify aTB or LTBI in young children.

[Screening for tuberculosis]. / Le dépistage de la tuberculose.

Rapid identification using bacteriological methods and adequate treatment of active tuberculosis cases are the most important objective of any tuberculosis activity but, in order to eliminate the disease, another important component of tuberculosis control is to reduce the vast reservoir of latent tuberculosis infections. Tuberculin skin test and interferon-gamma release assays are designed to identify immune response against mycobacterial antigens. Both tests are accurate to detect latent but not active forms of tuberculosis. Interferon-gamma release assays have higher specificity than tuberculin skin testing in BCG-vaccinated populations particularly if BCG was administered after 1 year of age. Both tests perform poorly to predict risk for progression to active tuberculosis. Screening should therefore be limited to situations with a clear likelihood of transmission after contact, taking account of the infectiousness of the index case and the intensity of exposure, or to those with a great probability of developing tuberculosis: young children and immunocompromised persons.

[Which immunizations for which adults in 2011?]. / Quelles vaccinations pour quels adultes en 2011?

Immunizations are extremely efficient in prevention of diseases with a lethal potential. Healthy adults, pregnant women and patients suffering from chronic diseases may have a different benefit from vaccine available in our country. Numerous health problems need to be addressed during a short consultation, relegating immunization to a position of secondary importance. This paper will address the issue of immunization in special circumstances such as: healthy adults, pregnant women, HIV-infected patients, patients with end-stage renal disease, patients with chronic liver diseases and solid organ transplant candidates and recipients.

[Pulmonary tuberculosis]. / La tuberculose pulmonaire.

Tuberculosis (TB) is a global health problem driven by poverty, HIV infection, etc. In Europe, the problem of multidrug resistance (i.e., resistance to at least rifampin and isoniazid) (MR) develops. The cases come essentially from the former U.S.S.R. In Belgium, the incidence of tuberculosis continues to decline to 9.4/100,000 inhabitants in 2008. The percentage of MR germs is 2.8%. The distribution of cases is not uniform across the country. The incidence is much higher among people recently coming from high prevalence countries than among the Belgian native. The pulmonary forms of TB are more contagious and more common. The clinical signs are frequently non specific. The diagnosis is often mentioned up after performing a chest Xray and must always be confirmed by microbiological examination and culture of several sputum or other respiratory specimens. It is very important to identify the germ, M. tuberculosis complex and to test its sensitivity to anti-TB agents. Standard treatment consists of 4 drugs: isoniazid, rifampin, ethambutol and pyrazinamide for 2 months followed by rifampin and isoniazid for at least 4 additional months. In suspected cases of MR, 5 drugs are prescribed at the outset. Treatment and duration will be adjusted according to the results of susceptibility testing. The potential toxicities of second-line drugs should be well known by the physicians. Compliance of the patient is essential. Screening in the entourage is part of the therapeutic process.

Dissection of cytotoxic and helper T cell responses.

Cytotoxic (CD8+) and helper (CD4+) T cells play a crucial role in resolving infections by intracellular pathogens. The development of technologies to visualize antigen-specific T cell responses in mice and men over the past decade has allowed a dissection of the formation of adaptive T cell immunity. This review gives a brief overview of the currently used detection techniques and possible future additions. Furthermore, we discuss our current understanding of the formation of antigen-specific T cell responses, with particular attention to the similarities and differences in CD4+ and CD8+ T cell responses, the functional heterogeneity within responder T cell pools and the regulation of CD8+ T cell responses by dendritic cells and CD4+ helper T cells.

Novel allogeneic granulocyte-macrophage colony-stimulating factor-secreting tumor vaccine for pancreatic cancer: a phase I trial of safety and immune activation.

PURPOSE: Allogeneic granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor vaccines can cure established tumors in the mouse, but their efficacy against human tumors is uncertain. We have developed a novel GM-CSF-secreting pancreatic tumor vaccine. To determine its safety and ability to induce antitumor immune responses, we conducted a phase I trial in patients with surgically resected adenocarcinoma of the pancreas. PATIENTS AND METHODS: Fourteen patients with stage 1, 2, or 3 pancreatic adenocarcinoma were enrolled. Eight weeks after pancreaticoduodenectomy, three patients received 1 x 10(7) vaccine cells, three patients received 5 x 10(7) vaccine cells, three patients received 10 x 10(7) vaccine cells, and five patients received 50 x 10(7) vaccine cells. Twelve of 14 patients then went on to receive a 6-month course of adjuvant radiation and chemotherapy. One month after completing adjuvant treatment, six patients still in remission received up to three additional monthly vaccinations with the same vaccine dose that they had received originally. RESULTS: No dose-limiting toxicities were encountered. Vaccination induced increased delayed-type hypersensitivity (DTH) responses to autologous tumor cells in three patients who had received >or= 10 x 10(7) vaccine cells. These three patients also seemed to have had an increased disease-free survival time, remaining disease-free at least 25 months after diagnosis. CONCLUSION: Allogeneic GM-CSF-secreting tumor vaccines are safe in patients with pancreatic adenocarcinoma. This vaccine approach seems to induce dose-dependent systemic antitumor immunity as measured by increased postvaccination DTH responses against autologous tumors. Further clinical evaluation of this approach in patients with pancreatic cancer is warranted.

Chemotherapy does not nullify the ability of donor lymphocyte infusions to mediate graft-versus-host reactions.

Two patients with multiple myeloma in relapse after allogeneic BMT received donor lymphocyte infusions (DLI) but later required chemotherapy for treatment of myeloma-related complications. In both patients, recovery from chemotherapy-induced aplasia was accompanied by manifestations of graft-versus-host reactions. The first patient developed grade II acute GVHD and a complete remission which has lasted >22 months. The second patient developed grade III acute GVHD but died with co-existing GVHD and extensive extramedullary myeloma. These results demonstrate that chemotherapy does not nullify the ability of donor lymphocytes to mediate graft-versus-host reactions.

Predictive factors for peripheral-blood progenitor-cell collections using a single large-volume leukapheresis after cyclophosphamide and granulocyte-macrophage colony-stimulating factor mobilization.

PURPOSE: (1) To study the ability of mobilized peripheral-blood progenitor cells (PBPC) collected in a single large-volume leukapheresis performed on a predetermined date to accelerate engraftment after high-dose cyclophosphamide and thiotepa; (2) to establish the minimum dose of PBPC associated with early engraftment; and (3) to identify parameters predictive of collection of large numbers of PBPC. PATIENTS AND METHODS: Twenty-three patients with breast cancer received cyclophosphamide (4 g/m2) and granulocyte-macrophage colony-stimulating factor ([GM-CSF] 5 micrograms/kg/d x 15 days) for PBPC mobilization. A single leukapheresis was performed 15 days after cyclophosphamide administration. Then, patients received high-dose cyclophosphamide and thiotepa followed by reinfusion of PBPC and 4-hydroperoxycyclophosphamide (4HC)-purged bone marrow. PBPC concentration was measured in serial peripheral-blood samples and in the leukapheresis product. Correlation analysis between PBPC dose and engraftment and between leukapheresis yield and patient characteristics was attempted. RESULTS: A single leukapheresis processed a median 36 L (range, 24 to 46) blood and collected 5 x 10(6) CD34+ cells/kg (< 0.3 to 24) and 6.2 x 10(5) colony-forming units granulocyte-macrophage (CFU-GM)/kg (< 0.001 to 29). All sixteen patients (70%) reinfused with > or = 2.9 x 10(6) CD34+ cells/kg reached a level of greater than 1,000 leukocytes/microL by day 13 and greater than 50,000 platelets/microL by day 15. All of these patients had a percentage of peripheral-blood CD34+ cells > or = 0.5%, and all but one, a level of greater than 100,000 platelets/microL, on the day of leukapheresis. The bone marrow CD34+ cell percentage at study entry predicted the number of CD34+ cells collected after PBPC mobilization (R2 = .42, P = .002). All patients with > or = 2.5% bone marrow CD34+ cells experienced early engraftment. CONCLUSION: Reinfusion of PBPC collected in a single leukapheresis accelerates engraftment in the majority of patients. Pretreatment bone marrow CD34+ cell content determines PBPC mobilization capacity and may help select hematopoietic rescue strategies.

Large-volume leukapheresis using regional citrate anticoagulation to collect peripheral blood progenitor cells.

The use of peripheral blood progenitor cells (PBPC) for hematopoietic rescue after high-dose chemotherapy is limited by the number of leukaphereses required to collect an adequate number of hematopoietic progenitors. To optimize the collection of PBPC, we evaluated a single large-volume leukapheresis protocol with citrate anticoagulation. A group of 23 patients received cyclophosphamide (4 g/m2) and GM-CSF (5 micrograms/kg/day for 15 days) as PBPC mobilization, with a single outpatient 6 h leukapheresis performed on the COBE Spectra 15 days later. Citrate (0.190 mmol/ml) was infused at 1.2 ml/L of blood/minute with a whole blood to citrate ratio between 17:1 and 25:1. Calcium chloride (50 mM) was administered at a citrate to calcium molar ratio between 10:1 and 5:1 to prevent hypocalcemia. A median 36.6 L (range 24.4-46.4) blood was processed using 338 mM citrate (269-473) and 50 mM calcium (25-75). A median 5 x 10(6) CD34+ cells/kg (< 0.3-24) and 6.2 x 10(5) CFU-GM/kg (< 0.001-29) were collected, representing 5.6 and 5.9 more PBPC, respectively, than were in circulation at the initiation of leukapheresis. We conclude that a 6 h large-volume leukapheresis following cyclophosphamide and GM-CSF mobilization is safe, can recruit hematopoietic progenitors into the circulatory compartment, and allows the collection of high numbers of PBPC in a single procedure.

Regulation of the T cell receptor-alpha mRNA expression in the human lymphoblastic T cell line CEM.

We analyzed the transcriptional events involved in the T cell receptor (TcR)-alpha mRNA expression in a human lymphoblastic T-cell line CEM. CD3-negative and CD3-positive CEM subclones that either lack mature TcR-alpha mRNA or express TcR-alpha mRNA were used. Exposure of the TcR-alpha mRNA negative subclones to phorbol 12-myristate 13-acetate (PMA) was followed by 2- to 3-fold increase of transcription, indicating that PMA acts on a transcriptional level. No increase of transcription was observed after blocking protein synthesis with cycloheximide (CHX) or after sequential stimulation with CHX followed by PMA. On the posttranscriptional level, CHX as well as PMA induced a progressive stabilization of TcR-alpha mRNA in the nuclear compartment, which was independent of ongoing transcription. The half-life of the TcR-alpha mRNA upon stimulation was about 6 hours. The accumulation of mature TcR-alpha mRNA seemed to be controlled by nuclear events on a transcriptional as well as posttranscriptional level. The data imply that alterations of TcR-alpha gene transcription are dependent on protein synthesis. DNA-binding proteins enhance transcription and labile nuclear proteins target TcR-alpha mRNA for rapid turnover.

Measurement of CD34+ cells in bone marrow by flow cytometry.

A procedure is described for the measurement of the %CD34+ progenitor cells in bone marrow using directly conjugated antibodies. Staining cells with anti-CD45.FITC in conjunction with anti-CD34.PE allows the CD45- nucleated red blood cells and the CD45++ lymphocytes and monocytes to be separated from the CD45+ progenitor cells. Granulocytes are separated from the CD34+ cells based on differences in side scatter properties. A gated acquisition of CD34+ cells is used to define the boundaries of the CD34+ population in a plot of forward scatter vs side scatter and in a plot of anti-CD45.FITC vs anti-CD34.PE. Use of these regions during analysis reduces background staining and allows for a consistent identification of a CD34+ population. Acquisition of 50,000 cells provides adequate precision of the %CD34+ measurement. Acquisition and analysis procedures are presented for use of both a Becton Dickinson FACScan flow cytometer and a Coulter EPICS Profile II flow cytometer.