T-cell bispecific antibodies in node-positive breast cancer: novel therapeutic avenue for MHC class I loss variants.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) represent a prognostic factor for survival in primary breast cancer (BC). Nonetheless, neoepitope load and TILs cytolytic activity are modest in BC, compromising the efficacy of immune-activating antibodies, which do not yet compete against immunogenic chemotherapy. PATIENTS AND METHODS: We analyzed by functional flow cytometry the immune dynamics of primary and metastatic axillary nodes [metastatic lymph nodes (mLN)] in early BC (EBC) after exposure to T-cell bispecific antibodies (TCB) bridging CD3ε and human epidermal growth factor receptor 2 (HER2) or Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5 (CEACAM5), before and after chemotherapy. Human leukocyte antigen (HLA) class I loss was assessed by whole exome sequencing and immunohistochemistry. One hundred primary BC, 64 surrounding 'healthy tissue' and 24 mLN-related parameters were analyzed. RESULTS: HLA loss of heterozygosity was observed in EBC, at a clonal and subclonal level and was associated with regulatory T cells and T-cell immunoglobulin and mucin-domain-3 expression restraining the immuno-stimulatory effects of neoadjuvant chemotherapy. TCB bridging CD3ε and HER2 or CEACAM5 could bypass major histocompatibility complex (MHC) class I loss, partially rescuing T-cell functions in mLN. CONCLUSION: TCB should be developed in BC to circumvent low MHC/peptide complexes.

Interleukin 10 induces apoptotic cell death of B-chronic lymphocytic leukemia cells.

Recent studies have established that interleukin (IL)-10 induces growth and most notably differentiation of normal human B lymphocytes. We studied here the effects of IL-10 on the proliferation and survival of B-chronic lymphocytic leukemia (B-CLL) cells. IL-10 was found to inhibit 54-96% of the spontaneous tritiated thymidine incorporation observed in 3 of 12 B-CLL samples. Furthermore, IL-10 decreased the viable cell recovery of all five B-CLL samples tested, irrespective of whether cells were spontaneously synthesizing DNA or not. After 1 wk, B-CLL populations cultured with IL-10 were lost while those cultured without IL-10 survived. Flow cytometric analysis, DNA gel electrophoresis, and Giemsa staining all revealed that IL-10 induced B-CLL cells to die from apoptosis. This IL-10-mediated apoptosis was dose dependent and specific as it could be inhibited by a neutralizing anti-IL-10 antibody. B-CLL cells undergoing apoptosis in response to IL-10 showed decreased Bcl-2 protein levels. Addition of IL-2, IL-4, interferon gamma, and anti-CD40 monoclonal antibody prevented the IL-10-mediated apoptosis of B-CLL cells. None of the malignant B cell populations obtained from eight non-Hodgkin's lymphomas and three hairy cell leukemias underwent apoptosis after IL-10 treatment, thus suggesting that the apoptotic effect of IL-10 is specific for B-CLL cells. Thus, IL-10 inhibits the DNA synthesis and most notably the survival of B-CLL cells, findings that call for considering IL-10 in the immunotherapy of chemoresistant B-CLL.

Interleukin 10 (IL-10) upregulates functional high affinity IL-2 receptors on normal and leukemic B lymphocytes.

Interleukin 10 (IL-10) has recently been shown to induce normal human B lymphocytes to proliferate and differentiate into immunoglobulin (Ig)-secreting cells. Herein, we show that IL-10 also promotes DNA synthesis and IgM production by anti-CD40 activated B cell chronic lymphocytic leukemia (B-CLL). Most strikingly, IL-2 and IL-10 were found to synergize to induce the proliferation and differentiation of B-CLL cells. This synergy between IL-2 and IL-10 was also observed with normal B cells which proliferated strongly and secreted large amounts of IgM, IgG, and IgA. The observed synergy is likely to be due to the IL-10-induced increase of high affinity IL-2 receptors on both normal and leukemic B cells. This increase of high affinity receptor is associated to an increase of Tac/CD25 expression that can be detected by flow cytometric analysis. Taken together, these results indicate that IL-10 permits anti-CD40 activated B cells to respond to IL-2 through an induction of high affinity IL-2 receptors. This effect of IL-10 may partly explain how T cells, which activate B cells in a CD40-dependent fashion, induce B cell proliferation and differentiation mostly through IL-2.

Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases.

Bruton's tyrosine kinase (BTK) is pivotal in B cell activation and development through its participation in the signaling pathways of multiple hematopoietic receptors. The mechanisms controlling BTK activation were studied here by examination of the biochemical consequences of an interaction between BTK and SRC family kinases. This interaction of BTK with SRC kinases transphosphorylated BTK on tyrosine at residue 551, which led to BTK activation. BTK then autophosphorylated at a second site. The same two sites were phosphorylated upon B cell antigen receptor cross-linking. The activated BTK was predominantly membrane-associated, which suggests that BTK integrates distinct receptor signals resulting in SRC kinase activation and BTK membrane targeting.

Inhibition of colon cancer growth by docosahexaenoic acid involves autocrine production of TNFα.

The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) has anti-inflammatory and anti-cancer properties. Among pro-inflammatory mediators, tumor necrosis factor α (TNFα) plays a paradoxical role in cancer biology with induction of cancer cell death or survival depending on the cellular context. The objective of the study was to evaluate the role of TNFα in DHA-mediated tumor growth inhibition and colon cancer cell death. The treatment of human colorectal cancer cells, HCT-116 and HCT-8 cells, with DHA triggered apoptosis in autocrine TNFα-dependent manner. We demonstrated that DHA-induced increased content of TNFα mRNA occurred through a post-transcriptional regulation via the down-regulation of microRNA-21 (miR-21) expression. Treatment with DHA led to nuclear accumulation of Foxo3a that bounds to the miR-21 promoter triggering its transcriptional repression. Moreover, inhibition of RIP1 kinase and AMP-activated protein kinase α reduced Foxo3a nuclear-cytoplasmic shuttling and subsequent increase of TNFα expression through a decrease of miR-21 expression in DHA-treated colon cancer cells. Finally, we were able to show in HCT-116 xenograft tumor-bearing nude mice that a DHA-enriched diet induced a decrease of human miR-21 expression and an increase of human TNFα mRNA expression limiting tumor growth in a cancer cell-derived TNFα dependent manner. Altogether, the present work highlights a novel mechanism for anti-cancer action of DHA involving colon cancer cell death mediated through autocrine action of TNFα.

In vitro Activation of B-CLL Cells.

Apart from surface Ig receptors, a variety of membrane molecules have now been described to deliver activation and progression signals to human B cells. Among them, CD40 antigen is likely to play a crucial role in the antigen-dependent maturation process. Recent studies performed in the laboratory have established that presentation of anti-CD40 mAbs in a crosslinked fashion by mouse Ltk(-) cells stably expressing human FcyRII/CDw32, allowed normal human B cells to enter into sustained proliferation. In their overwhelming majority, B-CLL cells are positive for CD40 expression. We have therefore examined the capacity of purified B-CLL cells to be stimulated by various cytokines for growth and differentiation, following crosslinking of slgs or CD40 antigen. In most B-CLL specimens studied, IL-2 was the sole factor, among a wide panel of cytokines tested, which reproducibly and significantly induced proliferation of leukemic B cells activated with anti-Ig reagents (SAC or anti-IgM antibodies). Unlike normal B cells, the great majority of anti-Ig activated B-CLL cells failed to proliferate in response to IL-4. In this activation system, IL-4 profoundly suppressed the IL-2 driven proliferation of B-CLL. An opposite pattern of growth-response was obtained following ligation of CD40 since IL-4 elicited proliferation of B-CLL whereas the growth-promoting effect of IL-2 was reduced. Under these culture conditions, IL-4 and IL-2 displayed additive effects on leukemic B cell growth. Surprisingly, IL-4 combined with anti-CD40 mAb allowed activation of certain leukemia specimens otherwise refractory to other stimulatory signals. Most B-CLL samples were induced for IgM synthesis upon SAC stimulation. This Ig response was potentiated by IL-2 and antagonized by IL-4. Anti-CD40 mAb used alone or in combination with cytokines (IL1-IL6, IFNγ, TNFα, TGFß) failed to induce Ig secretion from B-CLL. No evidence for Ig isotype switching was obtained with the cytokines listed above, whatever the mode of B-CLL activation. Taken together, our results suggest that B-CLL can be released in vitro from their apparent maturation block, by IL-2 and anti-Ig reagents or by IL-4 and immobilized anti-CD40 mAb. Additionally, the data reported here suggest that development of the agonistic and antagonistic activities of IL-4 on B cell growth and differentiation may depend upon the nature of the activation signal provided.

Experimental model for the investigation of kinetic and/or dynamic interactions between drugs and ethanol in humans.

This study was performed to establish an experimental method for the investigation of interactions between ethanol and drugs under predictable and controlled conditions. The model was tested with flumazenil (Ro 15-1788), a short-acting benzodiazepine antagonist with an elimination half-life of 1 h. Six healthy volunteers (5 males, 1 female) were administered ethanol by intravenous infusion with stepwise changing rates. The infusion rates were adapted to each subject on the basis of individual disposition parameters of ethanol, which were derived from preceding short-term infusions of 120 min duration (1.0 mg/kg in males, 0.8 mg/kg in the female). This two-step procedure led to individual ethanol plasma levels between 1.47 +/- 0.04 and 1.71 +/- 0.03 g/L, which were reached after 2.5 h and thereafter maintained over another 6 h. Within the period of constant ethanol levels, single doses of flumazenil and placebo, respectively, were injected intravenously as a bolus (2 min) in a double-blind fashion according to a randomized two-way crossover design. Three subjects received a dose of 0.10 mg/kg of flumazenil, and the remaining three subjects received a dose of 0.20 mg/kg. Evaluation of the plasma concentration time curves of flumazenil did not reveal evidence of an effect of ethanol on the pharmacokinetics of this drug.

[Mass screening in occupational health from the practitioner's viewpoint]. / Arbeitsmedizinische Reihenuntersuchungen aus der Sicht des Praktikers.

A practicing occupational physician presents his view of meaningful prophylactic examinations. Requirements for these examinations are summarized in 7 postulates: that priority be given to primary prevention, to efficiency, and to medical and occupational history taking, that conclusions from these examinations always be transformed into concrete measures at the workplace, that continuing bilateral exchange of information with the supervising governmental authorities be assured, that synergistic effects be taken advantage of for the goals of general preventive medicine, and that the collected data be evaluated epidemiologically.

Seasonal H1N1 2007 influenza virus infection is associated with elevated pre-exposure antibody titers to the 2009 pandemic influenza A (H1N1) virus.

The new influenza strain detected in humans in April 2009 has caused the first influenza pandemic of the 21st century. A cross-reactive antibody response, in which antibodies against seasonal H1N1 viruses neutralized the 2009 pandemic influenza A (H1N1) virus (2009 pH1N1), was detected among individuals aged >60 years. However, factors other than age associated with such a cross-reactive antibody response are poorly documented. Our objective was to examine factors potentially associated with elevated pre-exposure viro-neutralization and hemagglutination-inhibition antibody titers against the 2009 pH1N1. We also studied factors associated with antibody titers against the 2007 seasonal H1N1 virus. One hundred subjects participating in an influenza cohort were selected. Sera collected in 2008 were analysed using hemagglutination inhibition and viro-neutralization assays for the 2009 pH1N1 virus and the 2007 seasonal H1N1 virus. Viro-neutralization results were explored using a linear mixed-effect model and hemagglutination-inhibition results using linear-regression models for interval-censored data. Elevated antibody titers against 2009 pH1N1 were associated with seasonal 2007 H1N1 infection (viro-neutralization, p 0.006; hemagglutination-inhibition, p 0.018). Elevated antibody titers were also associated with age in the viro-neutralization assay (p <0.0001). Seasonal 2007 H1N1 infection is an independent predictor of elevated pre-exposure antibody titers against 2009 pH1N1 and may have contributed to lowering the burden of the 2009 pH1N1 pandemic.

[Long-term therapy of a metastasizing pancreatic vipoma using the somatostatin derivative octreotide]. / Langzeittherapie eines metastasierenden Pankreas-Vipoms mit dem Somatostatinderivat Octreotid.

A patient with metastatic VIP-producing pancreatic tumor was successfully treated with subcutaneous octreotide, an analogue of somatostatin, for more than 4 years. The profuse diarrhea was rapidly controlled and the plasma concentrations of the hormones (VIP, neurotensin, gastrin, pancreatic polypeptide) fell to nearly normal within 2 months. Because of asymptomatic increase in tumor size, we added chemotherapy 2 years later. Since the drug is rapidly effective and well tolerated, it will probably become the therapy of choice in this syndrome.

Lack of effect of the benzodiazepine antagonist flumazenil (Ro 15-1788) on the performance of healthy subjects during experimentally induced ethanol intoxication.

Flumazenil is a specific benzodiazepine antagonist. This study was designed to determine whether it also reverses CNS depression due to acute alcohol intoxication. Intoxication was experimentally induced in 6 healthy volunteers by intravenous infusion of ethanol. Individual constant ethanol plasma concentrations in the range 1.47 +/- 0.04 g.l-1 to 1.71 +/- 0.03 g.l-1 were maintained over 6 h. Two doses of flumazenil (0.1 or 0.2 mg.kg-1) and placebo were administered intravenously in a randomized, double-blind, two-way cross-over fashion. A battery of psychometric tests and subjective ratings of mood and performance were performed at baseline and at regular intervals during the study. Before the administration of flumazenil the characteristic symptoms and signs of ethanol intoxication were present in all subjects. Performance (measured by visual analogue scales), reaction time, digit symbol substitution test, and a tracing test, were markedly impaired by ethanol. After the injection of flumazenil three volunteers reported some subjective improvement in performance. However, in none of the subjects was there a difference between either dose of flumazenil and placebo in terms of an improvement in the objective psychometric variables.

Repair of common bile duct defects using the gallbladder or cystic duct as a pedicled graft.

To repair defects in the common bile duct, part of the gallbladder and the cystic duct may be used as a pedicled graft. This has the advantage of an independent blood supply and a related mucosal lining. The method is illustrated by four patients who had excellent primary and short term results. In two patients, the defect was due to large cholecystocholedochal fistulas caused by migrating large gallstones. In one patient with marked stenosis of the duct, the defect occurred when the stenosis was opened through a longitudinal incision. The fourth patient had a large duodenal ulcer that penetrated into the common duct, causing a defect that could not be closed by suture. A definite evaluation of the procedure must await a longer period of follow-up study.

[Diagnosis of pyloro-duodenal ulcer by means of radiology and endoscopy]. / Diagnostic de l'ulcère pyloro-duodénal par la radiologie et par l'endoscopie

Study of 100 cases of duodenal ulcer has revealed disagreement between radiologic and endoscopic findings. In 20 percent of cases the disagreement was minor (e.g. deformed duodenum - ulcer, deformed duodenum - scar, etc.). In 5 percent of cases they were major (e.g. normal - ulcer). The percentage of discrepancies, which were to the advantage of endoscopy, was lower than in the reports of JENNI and KAWAI, probably because our heterogeneous radiologic material was reviewed beforehand by two radiologists. The specific merit of endoscopy is that it makes it possible to observe superficial and sub-radiologic lesions of the mucosa, localize the ulcer with greater precision and detect deformation of the pylorus. Radiology affords better evidence of certain deformations.

Responsiveness of chronic lymphocytic leukemia B cells activated via surface Igs or CD40 to B-cell tropic factors.

Recent studies performed in the laboratory have established that interleukin-4 (IL-4) used in combination with anti-CD40 monoclonal antibody (MoAb) 89 presented on Ltk- mouse fibroblasts stably expressing human Fc gamma RII/CDw32 (referred to as the CD40 system) sustains long-term proliferation of normal human B cells. In the present study, B-cell chronic lymphocytic leukemias (B-CLLs) activated through slgs or CD40 were examined for their capacity to proliferate and differentiate in response to various cytokines. Our results indicate that the outcome of IL-4 stimulation on the in vitro growth of B-CLL depends on the signalling pathway used for their activation. Whereas IL-4 did not display any growth-stimulatory effect on B-CLL activated by Ig cross-linking agents, it could stimulate DNA synthesis and enhance the viable cell recovery when leukemic B cells were cultured in the CD40 system. Most B-CLL samples were induced for IgM synthesis upon Staphylococcus aureus strain Cowan I stimulation. This Ig response was potentiated by IL-2 and antagonized by IL-4. Anti-CD40 MoAb used alone or in combination with cytokines (IL-1 alpha to IL-6, interferon gamma, tumor necrosis factor gamma, and transforming growth factor beta) failed to induce Ig secretion from B-CLL cells. No evidence for Ig isotype switching was obtained with the cytokines listed above, regardless of the mode of activation. Taken together, our results suggest that B-CLL cells can be partially released from their apparent maturation block by IL-2 and Ig cross-linking agents. In contrast, combinations of IL-4 and cross-linked anti-CD40 antibodies induced entry of B-CLL cell into cycle, but poorly stimulated their differentiation into Ig secreting cells.

[Immunogenicity and stability of an aluminum-free liposomal hepatitis A vaccine (Epaxal Berna)]. / Immunogenität und Stabilität eines aluminiumfreien liposomalen Hepatitis-A-Impfstoffs (Epaxal Berna).

The high immunogenicity of the liposomal hepatitis A vaccine (Epaxal Berna) after a single dose and after a booster dose one year later has been confirmed in several studies with healthy adult volunteers: 95-100% and 96-100% seroconversion (> or = 20 mIE/ml) after 1 and 12 months respectively, as well as a booster effect in 100% of the cases after revaccination. The tolerability of this new, alum-free vaccine has been excellent with 6-25% local and 0-13% mild systemic reactions after a dose of 0.5 ml. Stability testing with and without detergent indicated partial internalization of the hepatitis A virions in the phospholipid bilayer of the liposome vesicles with storage. Immunization of 10 healthy adult volunteers with vaccine stored for 32 months at 4 degrees C showed, however, that the duration of storage has no influence on immunogenicity and tolerability of the vaccine.

Pain assessment after intramuscular injection.

Several parameters (pH, osmotic pressure) influencing the local tolerance of injectable drugs have been well-documented; however, little attention has been paid to pain following an injection--a common problem in clinical practice. A pain questionnaire was used to record pain up to 24 h after a deep ventrogluteal injection. Two groups of healthy volunteers were recruited: the first group (n = 6) received 3 different cotrimoxazole preparations and placebo and the second group (n = 10) received 4 different multivitamin preparations and placebo (double-blind, cross-over). Parameters monitored during and after injection included pain localization (line drawing), pain intensity (visual-analog scale: VAS) and verbal description of pain (pain rating index: PRI). In both groups, the equality of pain (VAS, PRI) induced by the preparations was rejected in all cases (Friedman's test, p < or = 1%). The pairwise comparisons of the groups showed significant differences (p < or = 5%) between various preparations. The correlation (Spearman's rank correlation) between pain parameters VAS and PRI was high. The present investigations have shown that the pain questionnaire is a valuable tool to investigate the subjective pain symptoms during and after the injection of different preparations.