Therapeutic Strategies and Clinical Outcome in Papillary Thyroid Microcarcinoma: A Multicenter Observational Study.

PURPOSE: Papillary thyroid microcarcinoma (MPTC) has an excellent prognosis. We aimed to evaluate the evolution of therapeutic strategies over time and the clinical outcome of MPTC. METHODS: In this retrospective multicenter observational study in a northwest Italian region, patients with intrathyroidal, unifocal tumor ≤1 cm in size, incidentally found at histology or preoperative cytology diagnosis, were included. Exclusion criteria were a previous head-and-neck irradiation and/or node metastases. RESULTS: From 1985 to 2012, 437 patients had an MPTC diagnosis, which was incidental in 85% and preoperative in 15%. Patients with a preoperative diagnosis were younger at the time of diagnosis (47.6 ± 12.7 years, p < 0.01) and had a larger tumor (7.0 ± 2.5 mm, p < 0.0001) than patients with an incidental diagnosis (age 52 ± 13.5 years, size 4.4 ± 2.8 mm), but there were no differences in clinical outcome between both groups. We observed a significant (p < 0.001) reduction in radioiodine remnant ablation during the years. TSH levels were: <0.1 mIU/l in 27.5%, 0.1-0.5 mlU/l in 33.7%, 0.5-2.5 mlU/l in 32.6%, 2.5-4.2 mlU/l in 3.9%, and >4.2 mlU/l in 2.3% of patients. Six patients (1.37%) had nodal recurrence; 5 of them were cured after therapy. MPTC-linked mortality was null. CONCLUSIONS: We confirmed the favorable clinical outcome of MPTC. Despite the reduction in radioiodine ablation, overtreatment of MPTC is still observed.

Antiflagellin antibodies recognize the autoantigens Toll-Like Receptor 5 and Pals 1-associated tight junction protein and induce monocytes activation and increased intestinal permeability in Crohn's disease.

BACKGROUND AND OBJECTIVES: Bacterial flagellin is considered an important antigen in Crohn's disease (CD) as it activates innate immunity through Toll-Like Receptor 5 (TLR5) engagement and induces an elevated adaptive immune response. Little is known about the presence of an autoimmune process in CD. We aimed to identify pathogenically relevant autoantigen targets in CD. METHODS: We screened a random peptide library with pooled sera of patients with active CD. Transepithelial flux of [3H] mannitol in T84 human intestinal epithelial cell line was used to study the epithelial barrier function. Monocyte activation was evaluated by surface expression of activation markers and by production of pro-inflammatory cytokines. Gene modulation of T84 cells exposed to antipeptide antibodies was analysed by gene array. RESULTS: We identified a peptide that shares homology with Salmonella typhimurium flagellin and with self-antigens such as TLR5 and cell junction protein, Pals 1-associated tight junction protein. The affinity-purified antipeptide antibodies recognized the self-antigens and induced increased intestinal epithelial cell permeability. Moreover, the antibodies induced monocyte activation upon binding TLR5. Finally, in cultured intestinal cells (T84) the purified antibodies induced the modulation of clusters of proinflammatory genes similar to the one induced by the engagement of TLR5 by its natural ligand flagellin. CONCLUSIONS: Antibodies directed against an immunodominant peptide of flagellin recognize self-antigens and are functionally active suggesting the presence of an autoimmune process that can both facilitate loss of tolerance to intestinal microflora by increasing cell permeability and amplify the innate immunity involvement through a novel mechanism of TLR5 activation.

CD1d expression on B-precursor acute lymphoblastic leukemia subsets with poor prognosis.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of childhood. Although therapeutical advances have been achieved, some ALL subgroups still fare poorly. CD1d is a monomorphic molecule that provides a suitable target for immunotherapy in view of the characterization of a glycolipid, alpha-galactosylceramide (alpha-GalCer), capable of being presented to CD1d-restricted T cells with cytotoxic potential. We investigated CD1d expression in 80 pediatric B-cell precursor (BCP) ALL cases defined according to immunophenotype, cytogenetic features and age at onset. CD1d was detected on ALL cells in 15% of the patients. CD1d+ ALLs were significantly associated with infant leukemia, pro-B phenotype and mixed-lineage leukemia (MLL)/AF4 gene rearrangement. Accordingly, overall survival of patients with CD1d+ ALL was significantly shorter. CD1d+ leukemic blasts were able to present alpha-GalCer via CD1d to cytotoxic CD1d-restricted T cells, which induced apoptosis of ALL cells that was inhibited by mAb to CD1d. CD1d+ blasts loaded with alpha-GalCer elicited cytokine secretion by CD1d-restricted T cells. Analysis of bone marrow (BM) cells derived from normal donors revealed that CD19+/CD1d+ cells were mostly mature B lymphocytes. However, a minority of BCPs expressed CD1d. Thus, expression of CD1d in ALL cases heralds an adverse prognosis but may provide a therapeutic tool.

Effect of cefoxitin and clindamycin on selection of derepressed mutants in Enterobacter cloacae.

The characteristics of an antibiotic that favor its ability to select for resistant bacteria are not completely understood. Otherwise, by the common use of broad-spectrum cephalosporins, resistant strains of several gram-negative species, especially Enterobacter cloacae, have been more frequently isolated. During our studies on beta-lactam resistance in E. cloacae, we observed that the addition of an inhibitor (clindamycin) to a potent inducer (cefoxitin) leads to an enhanced selection of resistant mutants. This could explain the emergence of beta-lactam resistant strains during antibiotic therapy.

Beta-lactamase production by Escherichia coli under different growth conditions.

Facultative anaerobic bacteria, such as Escherichia coli, are more resistant to cephalosporin antibiotics during anaerobic growth. Strict anaerobic ambience reduces beta-lactamase production or the enzyme affinities for their substrates. A different balance between DNA gyrase and topoisomerase I activity, during aerobic and anaerobic growth condition, could be related to the bacteria behavior.

CD85/LIR-1/ILT2 and CD152 (cytotoxic T lymphocyte antigen 4) inhibitory molecules down-regulate the cytolytic activity of human CD4+ T-cell clones specific for Mycobacterium tuberculosis.

Antigen-specific cytolytic CD4+ T lymphocytes control Mycobacterium tuberculosis infection by secreting cytokines and by killing macrophages that have phagocytosed the pathogen. However, lysis of the latter cells promotes microbial dissemination, and other macrophages engulf the released bacteria. Subsequently, CD4+ T-cell-mediated killing of macrophages goes on, and this persistent process may hamper control of infection, unless regulatory mechanisms maintain a subtle balance between lysis of macrophages by cytolytic CD4+ cells and activation of cytolytic CD4+ cells by infected macrophages. We asked whether inhibitory molecules expressed by CD4+ cytolytic T lymphocytes could play a role in such a balance. To this end, human CD4+ T-cell clones specific for M. tuberculosis were produced that displayed an autologous major histocompatibility complex class II-restricted lytic ability against purified protein derivative (PPD)-pulsed antigen-presenting cells. All T-cell clones expressed CD152 (cytotoxic T-lymphocyte antigen 4 [CTLA-4]) and CD85/leukocyte immunoglobulin-like receptor 1 (LIR-1)/immunoglobulin-like transcript 2 (ILT2) inhibitory receptors, but not CD94 and the killer inhibitory receptor (or killer immunoglobulin-like receptor [KIR]) p58.2. CD3-mediated activation of the clones was inhibited in a redirected killing assay in which CD152 and CD85/LIR-1/ILT2 were cross-linked. Specific antigen-mediated proliferation of the clones was also sharply reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked by specific monoclonal antibody (MAb) followed by goat anti-mouse antiserum. In contrast, blockade of the receptors by specific MAb only increased their proliferation. Production of interleukin 2 (IL-2) and gamma interferon (IFN-gamma) by the T-cell clones was also strongly reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked. The lytic activity of the T-cell clones against PPD-pulsed autologous monocytes or Epstein-Barr virus-activated B cells was increased by blockade and decreased by cross-linking of the receptors. These results indicate that CD152 and CD85/LIR-1/ILT2 play a role in the regulation of the antigen-specific activity of CD4+ cytolytic T lymphocytes against PPD-presenting cells.

Cytokine gene expression and T-cell proliferative responses in lymph node mononuclear cells from children with early stage human immunodeficiency virus infection.

BACKGROUND AND OBJECTIVES: The immunologic events taking place in secondary lymphoid tissue from children with early stage human immunodeficiency virus (HIV) infection are poorly understood. The aim of this study was to investigate cytokine gene expression and proliferative responses in lymph node (LN) biopsies from five children with early stage HIV infection, in the context of LN morphology and viral load. DESIGN AND METHODS: The design of the study was approved by the local Ethical Committee. Cytokine gene expression was studied in LN biopsies and in paired peripheral blood (PB) samples from HIV-infected children by reverse transcriptase-polymerase chain reaction. T-cell proliferation was assessed by 3H-thymidine incorporation. Viral burden in germinal centers was assessed by video densitometric analysis following immunohistochemical staining for HIV p24. RESULTS: Interleukin (IL)-2, IL-4 and IL-5 mRNA were not detected in any LN or PB sample from HIV-infected children. Interferon (IFN)-gamma mRNA was found only in CD8+ cells. IL-12 p35, IL-10, transforming growth factor-(TGF)-beta1, regulated on activation normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and IL-16 transcripts were detected in all samples. Proliferation of LN and PB mononuclear cells to polyclonal mitogens and soluble (recall and HIV-related) antigens was impaired as compared with the responses in a group of age-matched healthy controls. INTERPRETATION AND CONCLUSIONS: Changes in cytokine gene expression and T-cell proliferative responses are already detectable in lymph nodes from HIV-infected children at an early stage of disease.

The CD85/LIR-1/ILT2 inhibitory receptor is expressed by all human T lymphocytes and down-regulates their functions.

The inhibitory molecule CD85/LIR-1/ILT2 has been detected previously on the surface of a small proportion of T lymphocytes. In this study, evidence is provided that, although only a fraction of CD3+ cells are stained by mAb specific for CD85/LIR-1/ILT2 on their surface, this inhibitory receptor is present in the cytoplasm of all T lymphocytes, and that it is detectable on the surface of all T cell clones by the M402 mAb. Biochemical analyses further demonstrate that CD85/LIR-1/ILT2 is present in all T clones analyzed, and that the protein is tyrosine-phosphorylated. Expression of mRNA coding for CD85/LIR-1/ILT2 has been assessed by RT-PCR. Notably, in the NKL cell line and in one T cell clone, amplification of the messenger required 30 cycles only, whereas, in other T cell clones, an amplification product was detected by increasing the number of cycles. CD85/LIR-1/ILT2 inhibits CD3/TCR-mediated activation in both CD4+ and CD8+ clones, and it down-regulates Ag recognition by CD8+ cells in a clonally distributed fashion. Addition of anti-ILT2 HP-F1 mAb in the cytolytic assay enhances target cell lysis mediated by Ag-specific CTL. This could be due to interference of the mAb with receptor/ligand interactions. In contrast, HP-F1 mAb cross-linking triggers inhibitory signals that reduce cytotoxicity. CD85/LIR-1/ILT2 also controls responses to recall Ags and, in low responders, its engagement sharply increases T cell proliferation. The inhibitory function of the molecule is also confirmed by its ability to reduce CD3/TCR-induced intracellular Ca2+ mobilization.

Molecules that inhibit T-cell functions: cytochemical localization and shuttling.

Adaptive immune responses to antigens are mediated by specific receptors expressed on B cells (BCR's) and T cells (TCR's). Effector cells and memory cells are produced following a proliferative wave that accounts for clonal expansion. If not down-regulated, clonal expansion might lead to uncontrolled lymphoproliferation that would be harmful for the organism. Several mechanisms that account for the down-sizing of activated lymphocyte clones are briefly reviewed here. We next consider in detail one such mechanism that deals with the functional characterization and the immunocytochemical localization of two T-cell inhibitory molecules, namely the Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) and the HP-F1 antigen, both present in all T lymphocytes. CTLA-4 and HP-F1 inhibit CD4+ T-helper cell proliferation and the lytic ability of CD8+ T-cytotoxic cells in non-specific and in antigen-specific cytolytic assays. Interestingly, a clonal distribution exists as for the ability of CTLA-4 and HP-F1 to inhibit T-cell functions. In resting and activated T cells, both molecules are largely confined in the endosomal compartment, as shown by immunofluorescence analyses. However, upon interaction of T cells with Antigen-Presenting Cells (APC's) or with target cells that must be killed, CTLA-4 molecules are transported to the plasma membrane, at the site of cell-to-cell contact where, following interaction with ligands, they trigger inhibitory signals.

Cell activation via CD44 occurs in advanced stages of squamous cell carcinogenesis.

Squamous cell carcinoma (SCC) derives from dysplastic or metaplastic stratified epithelia. The process of squamous cell carcinogenesis has been investigated for the potential role of the adhesion molecule CD44, whose standard form (CD44s) and isoforms generated by alternative splicing of variant exons are known to display altered expression during tumorigenesis in other systems. We have utilized an in vitro correlate of squamous cell carcinogenesis, in which progression stages from normal squamous epithelium to dysplastic lesions and to SCC are represented by primary cultures of normal keratinocytes, by human papilloma virus-immortalized keratinocytes (UP) and by HPVimmortalized/v-Ha-ras transfected tumorigenic keratinocytes (UPR). We investigated expression of CD44 and of variant isoforms, from mRNA to intracellular and surface protein levels, and found no relationship between expression of CD44 and stages of squamous cell carcinogenesis. However, when the function of CD44 was analyzed as Ca(2+) mobilization ability upon monoclonal antibody binding and crosslinking, signal transduction via CD44 was found only for the neoplastic stage (UPR cells). Ca(2+) mobilization was completely independent of density of surface CD44. We have performed similar analyses in an in vitro model of SCC in which four squamous tumor cell lines and UPR cells were sorted according to increasing resistance to external cytotoxic stimuli, i.e. starving conditions, treatment with the retinoid N-(4-hydroxyphenyl)retinamide and cytolytic activity of effector lymphokine-activated killer cells. No relationship between expression of CD44 and level of cell resistance against external cell death-inducing stimuli was found, while CD44-mediated Ca(2+) mobilization ability was restricted to the highly resistant tumor cell lines. Our results indicate that the role(s) of CD44 in squamous cell proliferative disorders can be evinced from the functional features of the molecule, rather than from its phenotypic repertoire.

Natural analogue peptides of an HIV-1 GP120 T-helper epitope antagonize response of GP120-specific human CD4 T-cell clones.

Neutralizing antibodies and specific cytotoxic T lymphocytes (CTL) may contribute to controlling viral spread, and ideally, to virus clearance in HIV infection. Both effector mechanisms depend on specific CD4 T-helper (Th) cells. Nevertheless, HIV hypervariability facilitates appearance of escape mutants for antibodies and for CTL responses. Here we also show that natural mutations (i.e., from sequences of different HIV strains) in an immunodominant Th epitope recognized by human CD4 clones specific for the envelope glycoprotein gp120 escape CD4 T-cell recognition. Furthermore, several natural analogue peptides exert an antagonistic function by inhibiting proliferative response of T cells specific to gp120 with a wild-type sequence. If similar events occur in vivo, they may represent an additional escape mechanism for HIV. In fact, antagonism for CD4 Th response may occur during superinfection with a different strain, or with the appearance of a variant carrying a mutated antagonistic sequence. In both cases, impaired Th cell function could lead to reduced immune control of HIV infection by interfering with CTL and antibody response.

Antagonistic activity of HIV-1 T helper peptides flanked by an unrelated carrier protein.

Antagonism is the ability of a modified antigenic peptide (altered peptide ligand, APL) to prevent CD4 T cell activation by the original peptide. Here we show that antagonistic activity can be conferred to peptides of HIV envelope glycoprotein gp120 and reverse transcriptase p66 by adding flanking polypeptide sequences at the C or at the N terminus by genetic engineering, rather than by introducing substitutions by synthesis. The glutathione S-transferase (GST)-peptide system has been used to produce molecules that display the peptide at the appropriate end of the GST carrier. When the gp120 peptide 191-205 (pep24) was expressed at the C terminus of GST (GST-24), antigenicity of specific human CD4 T cells was maintained. In contrast, when the peptide was expressed at the N terminus of GST (24-GST), antigenicity was abolished and antagonistic activity was introduced. Similar results were obtained with a p66-derived peptide at the C terminus of the GST carrier. Antagonism was (1) specific; proliferation of a CD4 T cell line from the same donor responding to the envelope glycoprotein of another retrovirus, HTLV-1, was not affected; (2) reversible; proliferative response was rescued in T cells exposed to antigen-presenting cells (APC) pulsed with the antagonist; (3) dominant; T cells cultured with APC pulsed with the agonist and with APC pulsed with the antagonist did not proliferate. The carrier could be cleaved by proteolysis while the antagonistic activity was preserved. Thus a minimal sequence that confers antagonistic activity can be engineered or synthesized with peptides to antagonize undesired CD4 responses as an alternative to the use of APL.

CTLA-4 (CD152) inhibits the specific lysis mediated by human cytolytic T lymphocytes in a clonally distributed fashion.

Since the functional outcome of effector T lymphocytes depends on a balance between activatory and inhibitory receptors, we studied the ability of CTLA-4 (CD152) to inhibit the cytolytic function of CTL. In 22 TCR alpha/beta+ CD3+ 8+ CTL clones, activation induced by anti-CD3, anti-CD28, or anti-CD2 mAb was inhibited by anti-CD152 mAb in a redirected killing assay. In eight clones inhibition was >40%, in 10 it ranged between 20-40%, and in four it was <20%. This suggests the existence of a clonal heterogeneity as well as for the ability of CTLA-4 to inhibit CD3/TCR-, CD28-, or CD2-mediated CTL activation. To support further this contention, we used an experimental model based upon Ag-specific CTL. Eight Ag-specific T cell clones that lyse autologous EBV-infected B lymphocytes, but are unable to lyse allogeneic EBV-infected B cell lines, were used in a cytolytic assay in which anti-CD152 mAb or soluble recombinant receptor (i.e., CTLA-4 Ig) were included. In this system, at variance from the redirected killing assay, cross-linking of surface molecules by mAb does not occur. Thus, addition of anti-CD152 mAb or of CTLA-4 Ig and anti-CD80/CD86 mAb to the assay should result in a blockade of receptor/ligand interactions. As a consequence, inhibition of a negative signal, such as that delivered via CD152, should enhance lysis. A >40% increment of target cell lysis was achieved in three of eight clones studied. Since it is not equally shared by all CTL clones, this feature also appears to be clonally distributed.

Attenuated Listeria monocytogenes carrier strains can deliver an HIV-1 gp120 T helper epitope to MHC class II-restricted human CD4+ T cells.

Listeria monocytogenes is a facultative intracellular pathogen which, following uptake by macrophages, escapes from the phagosome and replicates in the cytoplasm. This property has been exploited using recombinant L. monocytogenes as a carrier for the intracytoplasmic expression of antigens when MHC class I-restricted cytotoxic T lymphocyte responses are required. Much less is known of the ability of these bacteria to trigger MHC class II-restricted responses. Here, we demonstrate that after ingestion of L. monocytogenes expressing a T helper epitope from the gp120 envelope glycoprotein of HIV, human adherent macrophages and dendritic cells can process and present the epitope to a specific CD4+ T cell line in the context of MHC class II molecules. No significant differences were observed when the attenuated strains were trapped in the phagolysosome or impaired in the capacity to spread intracellularly or from cell to cell. Similar results were obtained using carrier proteins that were either secreted, associated with the bacterial surface, or restricted to the bacterial cytoplasm. A dominant expression of the TCR Vbeta 22 gene subfamily was observed in specific T cell lines generated after stimulation with the recombinant strains or with soluble gp120. Our data show that in this in vitro system L. monocytogenes can efficiently deliver antigens to the MHC class II pathway, in addition to the well-established MHC class I pathway. The eukaryotic cell compartment in which the antigen is synthesized, and the mode of display seem to play a minor role in the overall efficiency of epitope processing and presentation.

Net effect of inoculum size on antimicrobial action of ampicillin-sulbactam: studies using an in vitro dynamic model.

To examine the predictable effect of inoculum size on the kinetics of the antimicrobial action of ampicillin-sulbactam, five TEM-1 beta-lactamase-producing Escherichia coli strains were studied in an in vitro dynamic model at two different initial inocula (N0S). All bacteria were exposed to ampicillin-sulbactam in a simulated system reflecting the pharmacokinetic profiles in human tissue after the administration of a single intravenous dose of ampicillin (2 g) plus sulbactam (1 g). Each strain was studied at low (4.0 to 5.2 log CFU/ml) and high (5.0 to 7.1 log CFU/ml) N0S. Despite pronounced differences in susceptibilities, the patterns of the killing curves observed with a given strain at different N0S were similar. As expected, viable bacterial counts increased with inoculum size. Striking visual contrasts in the respective curves for each organism were reflected by the area under the bacterial count-time curve (AUBC) but not by the difference between the N0 and the lowest bacterial counts (Nmin) at the nadir of the killing curve: the N0-associated changes in the AUBC on average were 75%, versus 2.5% for log N0--logNmin. To examine qualitative differences in antimicrobial effects at different N0S (i.e., the net effect of the inoculum), the difference in the high and low N0S was subtracted from each point on the killing curve obtained at the higher N0 for each strain. These adjusted curves were virtually superimposable on the observed killing curves obtained at the lower N0. Moreover, by using adjusted data, the AUBC values were similar at the two inocula, although slight (average, 11%) but systematic increases in the AUBC occurred at high N0S. Thus, there was only a weak net effect of inoculum size on the antibacterial effect of ampicillin-sulbactam. Due to similar slopes of the AUBC-log N0 plots, the antibacterial action at different N0S may be easily predicted by an approximate equation; the predicted AUBCs were unbiased and well correlated with the observed AUBCs (r = 0.997). Compiled data obtained with normalized AUBCs for different strains at different N0S yielded a positive correlation (r = 0.963) between the N0-normalized AUBC and the MIC of ampicillin-sulbactam. The adjustment and normalization procedure described might be a useful tool for revealing the net effect of the inoculum and to predict the inoculum effect if there are no qualitative differences in antimicrobial action at different inocula.

Anti-HIV genetic treatment of antigen-specific human CD4 lymphocytes for adoptive immunotherapy of opportunistic infections in AIDS.

HIV-1 infection results in the loss of CD4+ T helper lymphocytes which make up the immune repertoire. This leads to opportunistic infections that define AIDS. Here, we show that CD4 T cell lines from normal donors with specificity for different antigens can be rendered resistant to HIV-1 replication by retroviral transduction with an antisense vector directed to the HIV-1 tat gene. The genetic treatment did not affect the properties of antigen-specific CD4 lymphocytes such as proliferative response, lymphokine production and phenotypic markers. The HIV-1 challenge dose that resulted in productive infection was two to four logs higher for transduced cells as compared with control cells. Resistance was shown with the HXB2 strain, whose tat sequence was used to design the antisense gene, and with the SF2 strain, whose targeted tat sequence carries five nucleotide mismatches. Retroviral transduction was also performed on a Candida-specific T cell line from a seropositive individual. This line, derived from T cells infected in vivo, produced infectious virus when stimulated in vitro with antigen, but was no longer productive after transduction. In addition, a four log higher HIV-1 challenge dose was needed for a productive superinfection of this T cell line. The production of antigen-specific CD4 T cells resistant to HIV-1 replication to be used in adoptive immunotherapy of opportunistic infections may represent a new form of gene therapy of AIDS.

Antigenicity of HIV-derived T helper determinants in the context of carrier recombinant proteins: effect on T helper cell repertoire selection.

T helper (Th) epitopes can be included in a recombinant protein with B and CTL epitopes to create more effective immunogens. To determine whether the antigenicity of HIV Th epitopes is preserved in this altered molecular context, human Th clones specific for peptides of HIV gp120 and reverse transcriptase p66 were challenged with recombinant proteins carrying the antigenic epitopes in different sites. We found that a given epitope was recognized by a specific T cell clone only when it was inserted in a particular position of the carrier. However, the permissive position was not the same for all epitopes. Enzymatic excision from a nonpermissive context or insertion of a polyserine spacer between the epitope and the carrier restored antigenicity. Nevertheless, antigenicity was not abolished in a synthetic peptide encompassing the epitope and the neighboring residues from the nonpermissive location. These data suggest that, in this case, the primary sequence of the chimeric protein flanking the HIV peptide is not responsible for loss of antigenicity. Furthermore, constructs carrying the epitope in a given position were recognized by peptide-specific Th clones raised from some individuals, but not from others. We show that this is due neither to individual modes of processing nor to the use of distinct major histocompatibility complex MHC class II restriction elements, but rather that it is related to the fine specificity of the clones. To study the effect of epitope context on selection of T cell repertoire in a naive individual, T cell lines were generated in vitro by stimulation with different peptide constructs. This resulted in the induction of diverse clonotypes defined by the pattern of recognition of different constructs, by T cell receptor V beta gene usage and by fine epitope mapping.

Human T leukaemia virus type 1 (HTLV-1) specific T-helper cell response: clonal fluctuations and repertoire heterogeneity.

The naive T-helper (Th) repertoire specific for HTLV-1 envelope (env) has been examined on antigen specific T-cell lines and clones from non-immune individuals. Clonal heterogeneity was determined by analysing the T-cell receptor (TCR) Vbeta gene usage and by sequencing the hypervariable regions of the TCR genes. Fluctuations in the V beta gene usage were determined by comparing the TCR Vbeta gene profiles of T-cell lines at different times. We found that a diverse repertoire for HTLV-1 env could be triggered in vitro. Diverse Vbeta genes were used by the same line tested at different times, suggesting that clonal composition of an antigen-specific T-cell line is not constant in vitro. Clones in fact may be up- and down-regulated and clonotypes undetectable at one time point can emerge upon subsequent restimulation. Therefore evaluation of the clonal composition of a T-cell line gives a snapshot of the dominant clones at the time of analysis, and does not tell the whole picture of the antigen-specific ensemble. Furthermore, by sequencing the TCR genes, we identified clones with identical Vbeta gene usage which differed in hypervariable regions (CDR3), indicating their derivation from independent precursors and contributing to overall clonal heterogeneity. If these data can be extended to HTLV-1-infected patients studied in vivo, the Th cell repertoire specific for HTLV-1 env may prove very heterogenous, with important implications for vaccine development.

Predictors of effect of ampicillin-sulbactam against TEM-1 beta-lactamase-producing Escherichia coli in an in vitro dynamic model: enzyme activity versus MIC.

The clinical outcome in patients treated with ampicillin-sulbactam may not always be predictable by disc susceptibility testing or with the MIC as determined with a constant level (4 micrograms/ml) of the beta-lactamase inhibitor (MIC1). The enzyme activities (EA) and the MICs estimated at a constant ratio of ampicillin to sulbactam of 2:1 (MIC2) for 15 TEM-1 beta-lactamase-producing strains of Escherichia coli were examined as alternatives to MIC1 as predictors of the antibacterial effects of this combined drug as studied in an in vitro model which simulates ampicillin-sulbactam pharmacokinetic profiles observed in human peripheral tissues. Integral parameters describing the area under the bacterial count-time curve (AUBC), the area between the normal growth curve, and the killing curve of bacteria exposed to antibiotic (ABBC), and the second parameter expressed as a percentage of its maximal hypothetical value (ABBC/ABBCmax) were calculated. All three parameters correlated well with EA (AUBC, r = 0.93; ABBC, r = -0.88; ABBC/ABBCmax, r = -0.91) and with MIC2 (r = 0.94, -0.94, and -0.95, respectively) but not with MIC1. Both EA and MIC2 can be considered reliable predictors of the antibacterial effect of ampicillin-sulbactam in an in vitro model. These correlations suggest that in vitro kinetic-dynamic models might be useful to reexamine established susceptibility breakpoints obtained with data based on the MIC1 (MICs obtained with constant levels of beta-lactamase inhibitors). These data also suggest that quantitative determinations of bacterial beta-lactamase production and MICs based on the component concentration ratio observed in vivo might be useful predictors of the effect of ampicillin-sulbactam and other beta-lactam-inhibitor combinations.

Handling of retroviral antigens by human antigen-presenting cells.

Antigen-specific T helper cells play an important role in retroviral infections. Indeed, they provide help for B-cell activation and antibody production and for clonal expansion of cytolytic lymphocytes. Therefore, we used retrovirus-specific human T helper clones in order to define modes of antigen presentation, antigen-presenting cells and the molecular context of Th epitopes that could be exploited in the design of immunogens aimed at optimizing the Th cell response. In particular, we describe several mechanisms of receptor-mediated antigen uptake that enhance the stimulation of human T-cell clones specific for HIV and HTLV-1 antigens; we report on the differential recognition of Th epitopes depending on the molecular-viral context; we show that dendritic cells are the most efficient presenting cells and are essential for the induction of in vitro primary Th cell responses; and finally, we propose that Th cells specific for internal, conserved antigens of HIV such as reverse transcriptase, may be candidates for intrastructural help resulting in induction of envelope specific antibodies.