In situ delivery of allogeneic natural killer cell (NK) combined with Cetuximab in liver metastases of gastrointestinal carcinoma: A phase I clinical trial.

Despite successful introduction of NK-based cellular therapy in the treatment of myeloid leukemia, the potential use of NK alloreactivity in solid malignancies is still elusive. We performed a phase I clinical trial to assess the safety and efficacy of in situ delivery of allogeneic NK cells combined with cetuximab in liver metastasis of gastrointestinal origin. The conditioning chemotherapy was administrated before the allogeneic NK cells injection via hepatic artery. Three escalating doses were tested (3.106, 8.106 and 12.106 NK cells/kg) following by a high-dose interleukin-2 (IL-2). Cetuximab was administered intravenously every week for 7 weeks. Nine patients with liver metastases of colorectal or pancreatic cancers were included, three per dose level. Hepatic artery injection was successfully performed in all patients with no report of dose-limiting toxicity. Two patients had febrile aplasia requiring a short-term antibiotherapy. Grade 3/4 anemia and thrombopenia were also observed related to the chemotherapy. Objective clinical responses were documented in 3 patients and among them 2 occurred in patients injected with cell products harboring two KIR ligand mismatches and one in a patient with one KIR ligand mismatch. Immune monitoring revealed that most patients presented an increase but transient of IL-15 and IL-7 cytokines levels one week after chemotherapy. Furthermore, a high expansion of FoxP3+regulatory T cells and PD-1+ T cells was observed in all patients, related to IL-2 administration. Our results demonstrated that combining allogeneic NK cells transfer via intra-hepatic artery, cetuximab and a high-dose IL-2 is feasible, well tolerated and may result in clinical responses.

Quality controls on cord blood unit contiguous segments: recommendation of the SFGM-TC.

In the attempt to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapies (SFGM-TC) set up its fourth annual series of workshops which brought together practitioners from all of its member centers. These workshops took place in September 2013 in Lille. Literature and intra-laboratories studies suggest that attached segment is representative of cord blood unit (CBU). Nevertheless, some discrepancies have been observed when analyzing large data registries. To address these issues, we have listed recommendations to increase the standardization of segment processing and quality control (QC), information on units of measurement and specifications and action to be taken in case of out of specifications QC results on segment.

Diagnosis and management of nocardiosis after bone marrow stem cell transplantation in adults: lack of lymphocyte recovery as a major contributing factor.

Hematopoietic cell transplantation (HCT) is a curative treatment for hematological malignancies. This therapeutic approach is associated with a profound immune deficiency and an increased rate of opportunistic infections. Nocardiosis is a rare bacterial infection occurring mainly in patients with deficient cell-mediated immunity, such as AIDS patients or transplant recipients. Diagnosis of nocardiosis can be challenging, as signs and symptoms are non-specific. Routine prophylaxis with trimethoprin/sulfamethoxazole (TMP/SMZ) does not prevent the risk of infection. Between May 2001 and December 2009, five cases of nocardiosis were diagnosed from the 366 allogeneic HCT recipients in our centre. Four patients developed a disseminated nocardiosis within the first year after HCT. The fifth patient presented a localized cutaneous nocardiosis. In disseminated cases, median total CD4+ T-cells were below 100 cells/µL. Naive CD4+ CD45RA+/RO- T-cells were almost undetectable. CD8(+) T-cells and NK cells were below the normal range and CD19+ B-cell reconstitution was completely deficient. In a localized case, we observed a lack of naive thymic emigrants CD4+ CD45RA+/RO- T-cells.

[Quality control of defrosted cord blood units: results from an inter-laboratory study]. / Contrôle de qualité des greffons de sang placentaire décongelés : résultats d'une étude multicentrique.

PURPOSE: Today, haematopoietic stem cell graft from placental blood concerns more than 15 % of allogeneic grafts. An inter-laboratory study of the quality control of defrosted cord blood units has been coordinated by the French society for cell and tissue bioengineering (SFBCT), with the cord blood bank of Bourgogne Franche-Comté and controlled by the French health products safety agency (Afssaps). The aim of this study is to ensure the inter-laboratory reproducibility of the quality controls practised by the banks during defrosting. The cellular outputs were analyzed according to the defrosting techniques, according to the method used in flow cytometry: single-platform (SP) versus double-platform (DP), or the product nature, i.e. in total blood or miniaturized. METHODS: Forty-two units of placental blood (USP), which were out of range were provided for defrosting to 14 participating sites. USP were defrosted and controlled according to the procedures of each bank. Once the USP is defrosted, a part of the product was controlled by the site and the other part by Afssaps. Following controls were carried out: numeration of the total nucleated cells (TNC) and of CD34+ cells (made by a SP method in Afssaps) and functional assay. RESULTS: Concerning TNC, the defrosting sites obtained a cellular output of 94 %+/-28 in day 0 compared with an output of 72 %+/-24 in Afssaps showing a rather good stability of the USP transmitted with an average deviation of 23 %+/-22. The freezing process with or without reduction of volume does not affect this variation. Concerning the numeration of CD34+ cells, the average deviation between the participating sites and Afssaps was 29 %+/-23 compared with 21 %+/-16 for the sites using a SP method against 47 %+/-25 for those using a DP method. The CD34+ outputs are equal to 82 % +/- 60 in day 0 for the participating sites against 52 %+/-20 for Afssaps. For the sites using a DP method, it is stressed that this output is particularly high with a rate of 126 %+/-90 (n=15) whereas it is 62 %+/-20 (n=32) for the sites using a SP method. CONCLUSION: These results underline a good stability of viable CD34+ cells and a greater reliability of the SP methods for the CD34+ cell numeration for these defrosted USP. Lastly, the results of the functional assay regarding the average clonogenicities (equal to 15 %) reinforce the conclusions on the quality of the defrosted products.

[Extracorporeal photochemotherapy]. / La photochimiothérapie extracorporelle.

Photopheresis or extracorporeal photochemotherapy (ECP) is a cellular therapy which combines a leukapheresis followed by ex vivo treatment using psoralen and ultraviolet A irradiation before reinfusion into the patient. Its mechanisms of action remain unclear and selective photodestruction of leukocytes cannot explain the long-lasting immunomodulatory effects. Recent studies demonstrated that ECP down regulates the immune response and induces tolerance through the maturation of dendritic cells and the production of regulatory T cells. Based on these effects, ECP is mainly used for treatment of Sezary syndrome, graft-versus-host disease, organ graft rejection and autoimmune diseases. However, it is still not clear how ECP both activates tumor immunity against cutaneous T-cell lymphoma and induces tolerance in autoreactive disorders. In addition, the use of adjuvant therapies, the long-term effects and various treatment protocols remain to be investigated along with the specific indications.

[Banking and indications for cord blood grafts]. / Conservation et indications des greffons de sang placentaire.

A cord blood graft is now frequently used in the context of allogeneic hematopoietic transplantation. In 2007, 27% of unrelated allotransplantations performed in France used a cord blood graft. In comparison to other sources of hematopoietic grafts, cord blood has a number of advantages (immediate availability of the graft, possibility of a lesser HLA compatibility between donor and recipient...) and inconveniences (a limited number of stem cells, increased risk of infectious complications...). Recently, the possibility of combining two cord blood grafts has been demonstrated. In 2007, 55% of unrelated cord blood grafts transplantation were performed as such. Results of multicentric studies published as of now, as well as considerable potential associated with cord blood transplantation requires important efforts to increase both the quantity and quality of cord blood grafts made available for transplantation.

[Bacterial detection leading to pathogen inactivation]. / De la détection bactérienne à l'inactivation des pathogènes.

Bacterial contamination of blood components remains the highest infectious risk in blood transfusion, the risk is particularly high when it affects platelet concentrates (PC). In France, the residual risk of transfusion reaction due to bacterial contamination of PC has been decreasing slowly since 1994 but for all severity 1 case occurs with about 25,000 distributed PC and one death occurs with 200,000 distributed units. This reduction of the risk may be due to the measures which were implemented during the last 10 years in order to prevent contamination during donation. Improving strategies for reducing the risks of bacterial contamination is one of the priorities of the French National Blood Transfusion Service (l'Etablissement Français du sang - EFS). The main target remains PC. Bacterial detection or pathogens inactivation are now available and are able to reduce (for detection) or prevent (for inactivation) the occurrence of reaction due to bacterial contamination of PC. Up to now, the choice is in favour of bacterial detection. A national study was carried out in seven regional EFS at the end of 2004. It aims at confirming the feasibility of a systematic bacterial screening of PC before their delivery. The first conclusions show that this screening can be implemented with acceptable modifications in term of platelets availability. We can expect in a next future that new pathogens reduction technique and/or new detection systems will be available, certainly more efficient to prevent reaction due to bacterial contamination. Implementation of actual detection methods is probably a temporary solution.

[Blood transfusion and bacterial risk]. / Transfusion sanguine et risque bactérien.

Initial hemovigilance data confirm the incidence and severity of transfusion reactions due to the bacterial contamination of blood components (TRBC). With 18 deaths reported through the French hemovigilance network over the past 5 years, bacterial risks represent one of the major immediate complications of blood components (BC) transfusion. BC contamination may lead to more or less severe TRBC, depending on their origin: bacteria growth, the BC itself or unknown origin. Although the rate of donated blood or BC contamination is known (0.5% and 0.05%, respectively) it is still difficult to assess the actual incidence of TRBC, as it is difficult to identify them and relate them to transfusion. Likewise, better knowledge of bacteria, symptoms and outcome is required to improve prevention methods. Better prevention can reduce BC contamination and proliferation of bacteria at each stage of blood transfusion. Methods to detect BC contamination are still under investigation. Through continuous education of hemovigilance actors in identifying and dealing with TRBC, as well as drawing up procedures to perform inquiries and specific bacterial analyses, case reporting can be further improved in order to achieve more efficient prevention.

[Blood transfusion and bacterial risk]. / Transfusion sanguine et risque bactérien.

Initial hemovigilance data confirm the incidence and severity of transfusion reactions due to the bacterial contamination of blood components (TRBCs). With 18 deaths reported through the French hemovigilance network over the past five years, bacterial risks represent one of the major immediate complications of BC transfusion. BC contamination may lead to more or less severe TRBCs, depending on their origin: bacteria growth, the BC itself or unknown origin. Although the rate of donated blood or BC contamination is known (0.5% and 0.05%, respectively), it is still difficult to assess the actual incidence of TRBCs, as it is difficult to identify and relate them to transfusion. Likewise, a better knowledge of bacteria, symptoms, and outcome is required to improve prevention methods. Better prevention can reduce BC contamination and proliferation of bacteria at each stage of blood transfusion. Methods of detecting BC contamination are still under investigation. Through continuous education of hemovigilance participants in identifying and dealing with TRBCs, as well as drawing up procedures to perform inquiries and specific bacterial analyses, case reporting can be further improved, in order to achieve more efficient prevention.

Evaluation of the Gen-Probe amplified Mycobacterium tuberculosis direct test for the routine diagnosis of pulmonary tuberculosis.

A total of 624 respiratory specimens from 543 patients (418 Belgian, 110 Rwandan, and 15 Colombian patients) were tested for the presence of Mycobacterium tuberculosis by the Mycobacterium Tuberculosis Direct Test (MTDT, Gen-Probe). Compared to culture, the MTDT on 497 samples of sputum or broncho-alveolar lavage from Belgium had a sensitivity, specificity and positive and negative predictive value of 86.4%, 96.0%, 50.0% and 99.3% respectively. The pooled results for Rwanda (112 specimens) and Colombia (15 specimens) were 97.8%, 65.7%, 88.2%, 92% respectively. After resolution of discrepant results by taking into account the clinical data, the results for the Belgian patients were 86.9%, 96.2%, 52.6%, 99.3% respectively, and for the Rwandan-Colombian patients 98.1%, 100%, 100% and 92% respectively. Results could be improved by testing more than one specimen from each patient and the inclusion of an internal control to detect inhibitors of the reaction. Culture remains necessary for drug susceptibility tests and the isolation and identification of non-tuberculous mycobacteria.

[Pulmonary mycobacteriosis due to Mycobacterium xenopi" in-vitro sensitivity to classical antitubercular agents and clinical development]. / Mycobactériose pulmonaire à Mycobacterium xenopi: sensibilité "in vitro" aux antituberculeux classiques et evolution clinique.

Out of 11 patients suffering from Mycobacterium xenopi lung disease, 9 were treated with an empiric antituberculous triple chemotherapy until specific identification and antibiogram were available. Despite the important "in vitro" resistance to drugs, most of the patients improved; in the other patients, the impairment was always due to the underlying pathology. We conclude that the "in vivo" response of M. xenopi infections to antituberculous drugs is little influenced by the "in vitro" sensitivity.

Pulmonary tuberculosis in HIV-infected patients in Zaire. A controlled trial of treatment for either 6 or 12 months.

BACKGROUND: We studied the efficacy of a short-course regimen of chemotherapy for pulmonary tuberculosis in Kinshasa, Zaire. We also assessed whether, among patients with human immunodeficiency virus (HIV) infection, treatment should be extended from 6 to 12 months. METHODS: HIV-seropositive and HIV-seronegative outpatients with pulmonary tuberculosis were treated with rifampin, isoniazid, pyrazinamide, and ethambutol daily for two months, followed by rifampin plus isoniazid twice weekly for four months. The HIV-positive patients who had no evidence of tuberculosis were then randomly assigned to receive either rifampin plus isoniazid or placebo twice weekly for a further six months. We also followed a comparison group of HIV-seronegative patients who received no further treatment for tuberculosis after six months. RESULTS: After six months, 260 of 335 HIV-seropositive and 186 of 188 HIV-seronegative participants could be evaluated, and their rates of treatment failure were similar: 3.8 and 2.7 percent, respectively. At 24 months, the HIV-seropositive patients who received extended treatment had a relapse rate of 1.9 percent, as compared with 9 percent among the HIV-seropositive patients who received placebo for the second 6 months (P < 0.01). Extended treatment did not improve survival, however. Among the HIV-seronegative patients, 5.3 percent relapsed. CONCLUSIONS: Among HIV-seropositive patients with pulmonary tuberculosis, extending treatment from 6 to 12 months reduces the rate of relapse but does not improve survival. The six-month program of partly intermittent antituberculous treatment may be an acceptable alternative when resources are limited.

Faster identification of mycobacteria using gas liquid and thin layer chromatography.

Gas liquid chromatography (GLC) and thin layer chromatography (TLC) analysis of cell wall content was used for identification of mycobacteria isolated in primary cultures. GLC permitted determination of the fatty acid and alcohol profiles of Mycobacterium simiae and Mycobacterium marinum and detection of a peak in Mycobacterium ulcerans formerly described for Mycobacterium malmoense. Using the data obtained to fill some of the gaps in the dichotomic trees of Tisdall et al. and Jantzen et al., GLC analysis allowed full identification of 8 of 22 mycobacterial species after 24 hours. The other 14 species could be divided into four groups on the basis of similar findings on GLC. TLC was used for full identification of three species. The identification results of conventional methods were concordant with those of GLC and TLC in 161 of 169 strains (93%) representing 21 different species. Using primarily chromatography for analysis of cell wall content, and in the case of some species complementary biochemical tests, the identification procedure could be shortened to a maximum of three days after primary culture.

Anti-A60 immunoglobulin G in the serodiagnosis of tuberculosis in HIV-seropositive and seronegative patients.

OBJECTIVE: A60 is a high molecular weight mycobacterial antigen complex. The detection of immunoglobulin (Ig) G antibodies to A60 has been advocated as a reasonably sensitive and specific test for active tuberculosis (TB). We aimed to compare the sensitivity of this test among HIV-seropositive and HIV-seronegative patients with pulmonary TB. METHODS: The presence and concentration of anti-A60 IgG antibodies was assessed by enzyme-linked immunosorbent assay in 208 HIV-seropositive and 91 HIV-seronegative Zaïrian patients with smear-positive pulmonary TB. The relationship between anti-A60 IgG levels and HIV serostatus, CD4+ lymphocyte counts, presence of clinical AIDS, and tuberculin skin test results was verified. RESULTS: Only 36.5% of the HIV-seropositive, compared with 69.2% of the HIV-seronegative patients had a positive anti-A60 IgG test (P < 0.00001). Among HIV-seropositive patients, anti-A60 IgG levels did not differ according to CD4+ lymphocyte counts, presence of clinical AIDS, or tuberculin skin test results. CONCLUSIONS: Among patients with pulmonary TB, the sensitivity of testing for anti-A60 IgG was much lower among HIV-seropositive than among HIV-seronegative patients, even from the early stages of HIV-related immunodeficiency. This limits the utility of anti-A60 IgG-antibody testing in the diagnosis of TB among HIV-infected patients.

Spectrum of immunodeficiency in HIV-1-infected patients with pulmonary tuberculosis in Zaire.

Tuberculosis (TB) is the most common opportunistic infection in African patients who die from AIDS, yet the stage of immunodeficiency at which TB develops is uncertain. We studied the immune status of HIV-infected outpatients with pulmonary TB in relation to their clinical presentation in a cross-sectional study of 216 HIV-seropositive and 146 HIV-seronegative ambulatory incident cases of smear-positive and culture-positive pulmonary TB in Kinshasa, Zaire. HIV-seropositive and seronegative patients had median CD4 lymphocyte counts of 316.5/microL and 830.5/microL, respectively. Of the HIV-seropositive patients, 32.9% had less than 200 CD4 lymphocytes/microL, 37% between 200 and 499, and 30.1% 500 or more. Clinical AIDS, as defined by the WHO clinical case-definition or a modified version, was of similar limited use as a predictor of immunodeficiency. Among HIV-seropositive patients, oral candidosis, lymphopenia, a negative tuberculin purified protein derivative test, and cutaneous anergy were strongly associated with CD4 counts of less than 200/microL, and seemed to be better markers of immune dysfunction. We conclude that pulmonary TB develops across a broad spectrum of HIV-induced immunodeficiency and that a diagnosis of pulmonary TB is of limited use as a marker of stage of HIV disease in African HIV-infected outpatients.

PIP: Between March 1989 and September 1991, physicians compared CD4 lymphocyte counts of 216 HIV-seropositive patients whose sputum smears tested positive for pulmonary tuberculosis (TB) with those of 146 HIV- negative patients who also tested positive for TB at a TB screening center in Kinshasa, Zaire. The researchers wanted to investigate the immune status of HIV-infected outpatients with pulmonary TB in relation to clinical criteria. HIV seropositive patients had much lower CD4 lymphocyte counts than did HIV seronegative patients (total CD4 count, 316.5/mcl vs. 830.5/mcl; CD4 count, 13% vs. 36%; p .001). 90.4% of HIV-positive patients had CD4 counts 800 compared with 48% of HIV- negative patients. 32.9% of HIV-positive patients had CD4 counts 200 while just 1.4% of HIV-negative patients did. As CD4 counts fell, weight loss, diarrhea during the previous month, past or present herpes zoster, and oral candidosis occurred more frequently (p = .004 for oral candidosis and p = .02 for the rest). Negative purified protein derivative RT23 (PPD) tests and cutaneous anergy occurred more often as immunodeficiency rose (p .001). Increased immunosuppression was also characterized by no detectable cavitation on chest radiography, anemia, and low total lymphocyte counts (p = .02 for absence of cavitation and p .001 for the others). These results suggested that pulmonary TB occurred along the continuum of immunodeficiency as defined by CD4 counts. Thus, it is not likely to be a marker of the severity of HIV infection. Instead weight loss, diarrhea during the previous month, past or present herpes zoster, oral candidosis, negative PPD test and cutaneous anergy, absence of detectable cavitation on chest radiography, anemia, and low total lymphocyte appeared to be better markers of the severity of HIV-related immunodeficiency.

Three cases of opportunistic infection caused by propionic acid producing Corynebacterium minutissimum.

Propionic acid producing strains of Corynebacterium minutissimum were isolated from three patients with opportunistic infections. One neutropenic patient was undergoing chemotherapy for prolymphocytic leukemia; the other two patients were undergoing hemodialysis and peritoneal dialysis respectively. An unusual feature of these three strains was their resistance to several antibiotics, which is seldom seen in diphtheroids other than Corynebacterium jeikeium and CDC group D2.

[Nocardiasis. Apropos of a case report. Literature review]. / La nocardiose. A propos d'une observation. Revue de la litterature.

We report the observation of a cutaneous, pulmonary and osseous nocardiosis in a 45-year-old man. He was iatrogenously immunocompromised because of a "self-medication" with 32 mg per day of methylprednisolone during 30 months for gouty arthropathies. Under treatment with several antibiotics, a favourable evolution was obtained.