Micellar effects and analytical applications of nitro substitution in 4-Nitro-N-alkyl-1,8-naphthalimide by cysteine derivatives.

The aromatic nucleophilic substitution reactions of the nitro group of 4-Nitro-N-alkyl-1,8-naphthalimides by thiolate anions produce fluorescent derivatives and their rates are strongly accelerated by micelles of hexadecyltrimethylammonium chloride even at low pH. Acceleration factors of this reactions can reach million-fold. As the products are oxidant-insensible, this reaction allows the determination of SH- containing compounds such as cysteine, glutathione or proteins even in oxidative conditions. Limits of detection are as low as 5 × 10-7 M, ten times lower than the limit for the classic 5,5'-dithiobis-(2-nitrobenzoic) acid method. Moreover, this reaction can be developed at pHs between 6.5 and 7.5 thereby diminishing the rate of spontaneous oxidation of the thiols. In addition, we demonstrated that 4-Nitro-N-alkyl-1,8-naphthalimides can be used to evidence SH groups in peptides, proteins and living cells.

Utility of a Liquid Formulation of Levo-thyroxine in Differentiated Thyroid Cancer Patients.

The goal of levo-thyroxine (L-T4) administration in differentiated thyroid cancer (DTC) is to suppress thyroid stimulating hormone (TSH) levels. The tolerability and efficacy of a new formulation of liquid L-T4 vs. the previous tablet formulation was evaluated in a cohort of 59 patients with cured DTC. The correlation between breakfast modality and therapy was also monitored. Hormonal and clinical evaluations were performed before and 70 days after patients were switched from tablet to liquid L-T4 formulation, without changes in daily dose. Breakfast habits were evaluated. The interval between L-T4 therapy and breakfast was recorded. Patient approval of L-T4 formulations was evaluated. 8% of patients dropped out owing to adverse events. The modality of L-T4 administration proved adequate under tablet and liquid formulation in 64% and 68% of patients who fully complied with the protocol. While significantly more patients found the tablet formulation more agreeable, at the end of the protocol subjective symptoms had diminished significantly and 73% requested to remain on the liquid formulation. No change in TSH, thyroid hormones or thyroglobulin was noted during the study. A balanced breakfast containing less than 4 g of alimentary fibre did not interfere with L-T4 therapy. Liquid L-T4 seems to be a valid alternative formulation in DTC patients, its initial dislike being outweighed by a significant reduction in subjective symptoms. Both tablet and liquid L-T4 therapy require monitoring over time. A continental breakfast containing less than 4 g of alimentary fibres seems to favour the absorption of L-T4, whether in tablet or liquid formulation.

Efficacy of sorafenib and impact on cardiac function in patients with thyroid cancer: a retrospective analysis.

PURPOSE: Sorafenib has recently been recognized as an important standard option for the management of patients with differentiated thyroid cancer. Although data concerning cardiac safety are available in pan-tumor studies, no data are available on its use in everyday clinical practice in patients with thyroid cancer. METHODS: In the off-label program of our institution, we enrolled 14 patients with different histological types of thyroid cancer suitable for treatment with sorafenib. Our aims were to evaluate cardiac safety factors-LVEF (left ventricular ejection fraction), heart rate and blood pressure-the cardiac markers NT-proBNP and troponin I, radiological response evaluated by CT and (18)FDG-PET (according to RECIST 1.1 criteria) and biomarker reduction (Eastern Cooperative Oncology Group Performance Status: ECOG PS) 0-2. RESULTS: Patients with ECOG PS 2 accounted for 36%. After starting sorafenib, many patients displayed reduced or stabilized metabolic activity in target lesions (clinical benefit = 44%), radiologic reduction or stabilization (74%) and decreased cancer markers (90%). Lung metastases displayed the largest reductions in size. Median overall survival (OS) was 7 months and median progression-free survival (PFS) was 3 months. No sign of cardiotoxicity was observed in almost all patients. LVEF was altered in two patients and proved symptomatic in one. CONCLUSIONS: Sorafenib seems to be effective in reducing disease progression in the early stages of treatment (3-6 months). Responses varied considerably according to the criteria investigated. Cardiac toxicities did not raise concerns and were in line with data reported in other malignancies. However, cardiac monitoring is recommended.

Analysis of the antigen specific T cell repertoires in HIV infection.

In addition to HIV infection, several acquired immunodeficiencies lead to depletion of CD4 lymphocytes. These include immunosuppression resulting from high dose cancer chemotherapy or induced to control graft rejection, as well as in autoimmune diseases. The consequence of this depletion is an increased susceptibility to opportunistic infections or the inability to control primary infection in the case of HIV infection. In all instances a full or partial immunoreconstitution is desirable. In order to monitor the cellular immune state of a patient, rational information cannot be simply derived from phenotypic quantification of T lymphocytes. Instead loss or recovery of CD4 cells should be monitored by defining the specificity, the function and the clonality of the relevant cell population. Several methods are now available for this type of investigation. Here we describe an approach for the definition of clonal heterogeneity of antigen specific CD4 lymphocytes, a parameter that may help monitor loss or reconstitution in acquired immunodeficiencies. As examples of antigen specific CD4 T cell responses we focused on Pneumocystis carinii and on cytomegalovirus, as prototypic opportunistic pathogens which are responsible for severe infections in AIDS and in other immunosuppressive conditions which arise for instance following transplantation. Specific CD4 T cell lines were generated from normal controls and from seropositives in order to select antigen specific lymphocytes. The cells were subsequently analyzed for clonal diversity according to TCR BV gene family usage and according to TCR CDR3 size heterogeneity (spectratyping).

Temporal loss of Nef-epitope CTL recognition following macaque lipopeptide immunization and SIV challenge.

To address the subtle interactions between antiviral cytotoxic T-cell (CTL) immune responses and the evolution of viral quasispecies variants in vivo, we performed a longitudinal study in a simian immunodeficiency virus (SIV)-infected rhesus macaque that had a long experimental SIV infection before developing simian AIDS. Before being infected with SIV, this animal was immunized with a mixture of seven lipopeptides derived from SIV Nef and Gag proteins and showed a bispecific antiviral CTL response directed toward Nef 169-178 and 211-225 peptides. After SIV infection, CTL activity against the Nef 169-178 epitope was no longer detectable, as assessed from peripheral blood mononuclear cells stimulated by autologous SIV. CTL activity against the 211-225 epitope was lost after 3 months, and an additional CTL response to the amino acids 112-119 Nef epitope emerged. Analysis of the Nef proviral sequence revealed the presence of immune escape variants first in the 211-225 epitope and much later in the 112-119 epitope. In contrast, epitope 169-178 showed only two mutations among all viral sequencing performed. We conclude that in this macaque, bispecific CTL exerted a strong selective pressure and escape virus mutants finally emerged. We identified CTL recognizing a conserved Nef epitope 112-119 (SYKLAIDM), essential for viral replication, which could be associated with a prolonged AIDS-free period. These results stress the importance of the induction of broader multispecific CTLs directed against highly conserved and functional T-cell epitopes by vaccination, with the aim of keeping HIV infection in check.

Type 1 CD4(+) T-cell help is required for induction of antipeptide multispecific cytotoxic T lymphocytes by a lipopeptidic vaccine in rhesus macaques.

We have optimized the induction of antiviral cytotoxic T lymphocytes (CTL) in rhesus macaques by a lipopeptide vaccine containing seven peptides from simian immunodeficiency virus (SIV) Nef and Gag proteins and a strong T-helper peptide from tetanus toxoid (TT) that is promiscuous in humans (peptide TT 830-846). Two of the eight immunized macaques showed T-helper (Th) cell proliferation and a specific synthesis of gamma interferon in response to TT 830-846 peptide. They also showed multispecific cytotoxic activity against three to five of the immunizing SIV peptides. These results show the importance of a strong specific type 1 Th response for inducing a multispecific CTL response in vivo, which is essential for the development of an anti-human immunodeficiency virus vaccine.

Positive role of macaque cytotoxic T lymphocytes during SIV infection: decrease of cellular viremia and increase of asymptomatic clinical period.

We have measured cellular viremia and observed clinical outcome of macaques from two cohorts, the first including 12 macaques infected by SIVmac251 and the second including 12 macaques immunized by lipopeptides and then challenged by SIVmac251. In the first cohort (SIV-infected macaques), 3 patterns of CTL responders were determined: high, low and non-responders. In the macaques belonging to pattern of low and non-responders, cellular viremia, measured by growing the virus from PBMC, was continuously high during the first 6 months after infection, and five macaques developed AIDS within 14.4 +/- 7.7 months. Conversely, in the six high-responder macaques, cellular viremia was constantly low and only one macaque developed AIDS at 19 months, the five others being alive at 24 months. After immunization with lipopeptides, 7/12 macaques showed CTL responses and among these, after SIV challenge, cellular viremia was continually low, and no disease was observed at 22 months of follow-up. Conversely, the five non-responder macaques displayed persistent high viremia and macaques developed AIDS within 12.6 +/- 2.9 months after SIV challenge. These data strongly suggest that the presence of cytotoxic responses is inversely correlated with cellular viremia and correlated with overall survival and thus in an important component of the immune response in vaccinated individuals. It supports the idea that a strengthening of the CTL responses, if possible, might be beneficial in HIV-infected human beings.

Recognition of human T-leukemia virus (HTLV-1) envelope by human CD4+ T-cell lines from HTLV-1 seronegative individuals: specificity and clonal heterogeneity.

Because T-helper cells are critical for immune responses in retroviral infections, CD4+ T-cell lines specific for the human T-leukemia virus type 1 (HTLV-1) envelope have been generated from peripheral T lymphocytes of nonimmune donors to study their naive repertoire. Recombinant fragments (RE1, amino acids [aa] 26-200; RE3, aa 165-307; RE5, aa 308-401; and RE6, aa 165-401) of HTLV-1 envelope, whole envelope glycoprotein, and synthetic peptides were used to induce T-cell lines. CD4+ T-cell lines specific for one or more fragments were obtained from seven of eight individuals tested. T-cell lines generated against envelope glycoprotein from five of five donors did not cross-react with the RE fragments and vice versa. The lines specific for RE and env were mapped with overlapping peptides. The lines with single peptide (narrow) specificity contained a variety of clones that used different T-cell receptor V beta genes. These data (1) suggest that most of the normal individuals carry T-helper precursors specific for epitopes on HTLV-1 envelope; (2) indicate that heterogeneity of HTLV-1 envelope-specific T cells can be detected in the naive repertoire; and (3) define optimal antigenic preparations to be used to assess cellular immunity in HTLV-1-infected individuals.