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.

Immunomodulatory effect of vitamin D and its potential role in the prevention and treatment of thyroid autoimmunity: a narrative review.

The main role of vitamin D is to control mineral homeostasis. However, recent studies suggested the existence of a number of extraskeletal effects. Among the latter, preclinical studies provided consistent data on the involvement of vitamin D in innate and adaptive immunity and autoimmunity. Molecular biology studies showed that both vitamin D receptor and vitamin D enzymatic complexes are expressed in a large number of cells and tissues unrelated to mineral homeostasis. In contrast, only a few randomized clinical trials in humans investigated the possible role of vitamin D in the prevention or treatment of immunological disorders. In this regard, low serum vitamin D levels have been reported in observational trials in human autoimmune disorders. The aim of the present paper was to review the potential implications of vitamin D in immune modulation, with special focus on thyroid autoimmune disorders.

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.

Long-term outcome after radioiodine therapy with adjuvant rhTSH treatment: comparison between patients with non-toxic and pre-toxic large multinodular goitre.

In multinodular goitre (MNG), low radioiodine (RAI) activity after recombinant human (rh) TSH is able to reduce thyroid volume (TV) and improve symptoms. Our aim was to evaluate the long-term outcome of RAI after rhTSH treatment in patients who were divided according to their baseline TSH levels. Eighteen patients (69.2 ± 6.1 year) presented non-toxic (TSH >0.3 mIU/l) MNG (TV: 61.0 ± 3.8 ml; group 1), while 13 patients (74.1 ± 7.9 year) had non-autoimmune pre-toxic (TSH <0.3 mIU/l) MNG (TV: 82.6 ± 14.4 ml; group 2). TSH, thyroid hormones, TV (by ultrasonography), body mass index (BMI), symptoms and quality of life (QoL) were evaluated. Treatment induced short-term thyrotoxicosis in both groups, but this was slightly more marked in group 2 than in group 1. The number and severity of adverse events were similar. The follow-up period was 55.3 ± 4.1 months in group 1 and 57.2 ± 5.1 months in group 2. The final TV reduction was similar in groups 1 (63.4 ± 3.6%) and 2 (57.2 ± 4.6%) and TV reduction positively correlated only with initial TV. At the last examination, 14 group-1 subjects were on L-T4 therapy, while 2 group-2 subjects were on methimazole. An increase in BMI was noted only in group 2. MNG-related symptoms were significantly reduced in both groups. Symptoms related to sub-clinical hyperthyroidism improved in group 2, while no significant changes in QoL were noted in either group. This study confirms the effectiveness of rhTSH adjuvant treatment in reducing TV after low RAI activities, irrespective of baseline thyroid status. TSH levels <0.3 mIU/l proved to be predictive of a more severe thyrotoxic phase after rhTSH and RAI, while initial TSH levels >0.3 mIU/l were more frequently followed by a need for L-T4 therapy. Compressive symptoms improved in the majority of subjects.

Down syndrome, autoimmunity and T regulatory cells.

Autoimmune diseases are more represented in Down syndrome (DS) individuals compared to chromosomally normal people. Natural T regulatory cells (nT(reg) ) have been considered to be primary in the role of controlling the intensity and targets of the immune response. We have investigated the phenotypical and functional alteration of nT(reg) in a group of DS people. The phenotypical characteristic of T(reg) cells of 29 DS was analysed and compared with an age-matched healthy control group. The inhibitory potential of CD4(+) CD25(high) CD127(low) T regulatory cells was evaluated on autologous CD4(+) CD25(-) T cell proliferation in response to activation with a mytogenic pan-stimulus (anti-CD2, anti-CD3 and anti-CD28 antibodies). The CD4(+) CD25(high) cells in the DS and control groups were 2·692±0·3808%, n=29 and 1·246±0·119, n=29%, respectively (P=0.0007), with a percentage of forkhead box protein 3 (FoxP3)-expressing cells of 79·21±3·376%, n=29 and 59·75±4·496%, respectively (P=0.0015). CD4(+) CD25(+) FoxP3(+) cells were increased in peripheral blood from DS subjects (DS mean 5·231±0·6065% n=29, control mean 3·076±0·3140% n=29). The majority of CD4(+) CD25(high) were CD127(low) and expressed a high percentage of FoxP3 (natural T(reg) phenotype). While the proliferative capacity of DS T cells was not altered significantly compared to normal individuals, a reduced inhibitory potential of T(reg) compared to healthy controls was clearly observed (mean healthy control inhibition in T(eff) : T(reg) 1:1 co-culture: 58·9%±4·157%, n=10 versus mean DS inhibition in T(eff) :T(reg) 1:1 co-culture: 39·8±4·788%, n=10, P=0.0075; mean healthy control inhibition in T(eff) : T(reg) 1:0·5 co-culture: 45·10±5·858%, n=10 versus DS inhibition in T(eff) : T(reg) 1:0·5 co-culture: 24·10±5·517%, n=10, P=0.0177). DS people present an over-expressed peripheral nT(reg) population with a defective inhibitory activity that may partially explain the increased frequency of autoimmune disease.

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.

Recognition of antigenic clusters of Candida albicans by T lymphocytes from human immunodeficiency virus-infected persons.

The fine specificity of the cellular immune response to Candida albicans (i.e., recognition of different antigenic components) between normal controls and human immunodeficiency virus-infected patients in various stages of disease was compared. C. albicans-specific T cells, enriched by antigen stimulation and interleukin-2 expansion, were challenged with antigenic fractions of different molecular weight obtained by SDS-gel fractionation of C. albicans extracts in the presence of autologous mononuclear cells as antigen-presenting cells. Proliferative responses showed similar patterns of reactivity between controls and category A and B seropositive subjects. Category C patients with concurrent C. albicans infections did not give rise to C. albicans-specific T cell lines, confirming the T cell defect. Patients without clinically evident C. albicans infection had a low but broad reactivity pattern of C. albicans-specific T cells. These results suggest that depletion of C. albicans-specific T cells, independent of their fine specificity, occurs along with disease progression.

Selection of virus variants and emergence of virus escape mutants after immunization with an epitope vaccine.

In this report, we assessed the evolution of the cytotoxic T-lymphocyte (CTL) response induced by an epitope vaccine. In two macaques immunized with a mixture of lipopeptides derived from simian immunodeficiency virus (SIV) Nef and Gag proteins, CTL responses were directed against the same, single epitope of the Nef protein (amino acids 128 to 137) presenting an alanine at position 136 (Nef 128-137/136A). However, after 5 months of SIV infection, peripheral blood mononuclear cells from both macaques lost their ability to be stimulated by autologous SIV-infected cells while still retaining their capacity to generate cytotoxic responses after specific Nef 128-137/136A peptide stimulation. The sequences of the pathogenic viral isolate used for the challenge showed a mixture of several variants. Within the Nef epitopic sequence from amino acids 128 to 137, 82% of viral variants expressed the epitopic peptide Nef 128-137/136A; the remaining variants presented a threonine at position 136 (Nef 128-137/136T). In contrast, sequence analysis of cloned proviral DNA obtained from both macaques 5 months after SIV challenge showed a different pattern of quasi-species variants; 100% of clones presented a threonine at position 136 (Nef 128-137/136T), suggesting the disappearance of viral variants with an alanine at this position under antiviral pressure exerted by Nef 128-137/136A-specific CTLs. In addition, 12 months after SIV challenge, six of eight clones from one macaque presented a glutamic acid at position 131 (Nef 128-137/131E+136T), which was not found in the infecting isolate. Furthermore, CTLs generated very early after SIV challenge were able to lyse cells sensitized with the Nef 128-137/136A epitope. In contrast, lysis was significantly less effective or even did not occur when either the selected peptide Nef 128-137/136T or the escape variant peptide Nef 128-137/131E+136T was used in a target cell sensitization assay. Dose analysis of peptides used to sensitize target cells as well as a major histocompatibility complex (MHC)-peptide stability assay suggested that the selected peptide Nef 128-137/136T has an altered capacity to bind to the MHC. These data suggest that CTL pressure leads to the selection of viral variants and to the emergence of escape mutants and supports the fact that immunization should elicit broad CTL responses.

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.

In vitro determination of optimal antifungal combinations against Cryptococcus neoformans and Candida albicans.

There is currently no rapid, reliable, and reproducible in vitro technique to describe the growth-inhibitory interactions of antifungal drug combinations over a wide range of drug concentrations. We have developed a microdilution plate assay that was used to determine optimal drug combinations and concentrations of one-, two-, and three-drug regimens of amphotericin B (AmphB), fluconazole (FLU), and 5-fluorocytosine (5FC) for growth inhibition of three isolates each of Cryptococcus neoformans and Candida albicans. These growth inhibition data were then used in a multifactorial design technique to (i) generate contour and surface response plots to aid visual interpretation and (ii) develop mathematical equations describing the growth responses of the fungi to a wide range of antifungal concentrations and ratios. Our data indicated that (i) antifungal drug-drug interactions affecting yeast growth are complex functions of the drugs used in combination, their absolute concentrations, and also their relative (proportional) concentrations; (ii) AmphB-FLU combinations had additive effects against C. albicans over wide concentration ranges for each agent but were indifferent (i.e., were less than additive) in their inhibitory effect on C. neoformans; (iii) other two-drug combinations (FLU-5FC or AmphB-5FC) had indifferent effects on the growth of both fungi; and (iv) three-drug combinations (AmphB-FLU-5FC) showed an additive inhibitory effect on the growth of both C. albicans and C. neoformans. The finding that no antagonism was observed in combinations employing AmphB and FLU in this in vitro model is of critical importance since it argues against the current theoretical concept, based on the individual drug's mode of action, of antagonism between these two drugs. These microdilution techniques provide a method to determine rational regimens of antifungal agents in multidrug combinations for future testing to correlate in vitro activity with in vivo response. The use of this approach has made the evaluation of complex antifungal drug-drug interactions possible and provided important new information to the evolving field of antifungal drug combination.

Human CD4+ T cells can discriminate the molecular and structural context of T epitopes of HIV gp120 and HIV p66.

CD4+ T cell lines and clones specific for human immunodeficiency virus (HIV) antigens have been generated from peripheral lymphocytes of naive individuals by priming with the envelope protein gp120, the enzyme reverse transcriptase (p66), and their synthetic peptides. T cells were tested for proliferation to proteins, to peptides, and to HIV virions. Different patterns of reaction were identified. T cells primed in vitro with the whole antigen responded to the protein, but recognition of overlapping peptides occurred with a fraction of the lines or clones. The virus was recognized by some, but not all, of the gp120- and p66-specific T cells, with an efficiency 2 logs higher than the recombinant soluble proteins on a molar basis. One T cell line specific for gp120 responded to virions presented by B cells, but not by monocytes. In contrast, T cells induced with peptides did not always respond to the proteins. Generation of T cell lines from naive individuals may be an in vitro model for T cell immunization, and the response patterns may have implications for the design of vaccines aimed at inducing a T helper response. In fact our in vitro data suggest that (a) immunization with peptides does not always induce T cells recognizing the whole protein, (b) immunization with proteins does not always induce T cells recognizing the protein in the context of the HIV virus, and (c) recognition of gp120 in the context of HIV may be dictated by the type of presenting cells.

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.

Staphylococcus aureus bacteremia and endocarditis. New diagnostic and therapeutic concepts.

Staphylococcus aureus is an important and prevalent cause of both uncomplicated bacteremia, as well as endocarditis. This article addresses the following controversial strategies: diagnosis of endocarditis in staphylococcemic populations; optimal treatment strategies for right-sided and left-sided S. aureus endocarditis; use of echocardiography in S. aureus endocarditis; and therapy options for catheter-associated staphylococcemias.