Effect of interferon-alpha therapy on epitope-specific cytotoxic T lymphocyte responses in hepatitis C virus-infected individuals.

The majority of hepatitis C virus (HCV)-infected individuals fail to resolve the infection and become chronically infected despite the presence of HCV-specific CTL responses directed to different HCV-derived peptide antigens. Only a minority of individuals is able to clear the virus by mounting efficient CTL responses early after acute infection, but at present it is not clear whether viral clearance is associated with CTL responses of defined specificity. To elucidate those responses associated with improvement of the disease, we analyzed CTL responses to 16 different HLA-A2-presented, HCV-derived epitopes in 12 chronically infected patients, 14 chronically infected patients treated with interferon-alpha, and in one patient with acute symptomatic disease. We show here that the majority of chronically infected individuals present CTL responses directed to an NS4-derived peptide antigen (amino acids 1789-1797). Treated patients presented stronger HCV-specific CTL responses and therapy-induced changes in CTL target choice. In particular, 13 out of 14 individuals responded to an NS3-derived epitope (amino acids 1073-1081). By longitudinal analysis we show that five individuals responding to IFN-alpha therapy with decreases in alanine aminotransferase levels presented a strong CTL activity directed to the NS3-derived epitope. One patient that spontaneously resolved the infection presented a generally strong CTL activity specific for HCV-derived epitopes with a dominant response to the NS3-derived peptide antigen. This suggests that CTL responses directed to this NS3-derived antigen may be beneficial for the control of HCV infection. Improvement of these responses may represent a therapeutic intervention in chronic HCV infection.     

Plasminogen activator system modulates invasivecapacity and proliferation in prostatic tumor cells

The malignant phenotype of prostatic tumor cells correlates with the expression of both uPA and itscell-membrane receptor (uPAR); however, there is little information concerning the role of cell-bound uPAin matrix degradation and invasion. Our results suggest that cell-associated uPA plays a key role in regulat-ingthe amount of plasmin present at the surface of prostatic carcinoma (PRCA) cells and show that differ-entialproduction of uPA corresponds with the capacity to bind and activate plasminogen. In addition, weprovide direct evidence that both uPA secretion and the presence of uPA-uPAR complexes characterize theinvasive phenotype of PRCA cells and suggest the existence of several pathways by which tumor cells acquireplasmin activity. LNCaP cells (which do not produce uPA but express uPAR) may activate plasmin throughexogenous uPA. In vivo, the source of uPA may be infiltrating macrophages and/or fibroblasts as observedin several other systems. PAI-1 accumulation in the conditioned medium (CM) limits plasmin action to thepericellular microenvironment. Our results indicate that MMP-9 and MMP-2 are also activated by plasmingenerated by cell-bound but not by soluble, extracellular uPA. Plasmin activation and triggering of the pro-teolyticcascade involved in Matrigel invasion is blocked by antibodies against uPA (especially by anti- A-chainof uPA which interacts with uPAR) and by PA inhibitors such as p-aminobenzamidine which mayregulate levels of cell-bound uPA. uPA may also regulate growth in PRCA cells. Indeed, antibodies againstuPA A-chain (and also p-aminobenzamidine treatment) interfere with the ATF domain and inhibit cell growthin uPA-producing PC3 and DU145 prostate cancer cell lines, whereas exogenous uPA (HMW-uPA with ATF)induces growth of LNCaP prostate tumor cell line. These data support the hypothesis that in prostatic can-cerpatients at risk of progression, uPA/plasmin blockade may be of therapeutic value by blocking both growthof the primary tumor and dissemination of metastatic cells. ©Kluwer Academic Publishers     

Tolerance in TCR/Cognate Antigen Double-Transgenic Mice Mediated by Incomplete Thymic Deletion and Peripheral Receptor Downregulation

Influenza nucleoprotein (NP)-specific T-cell receptor transgenic mice (F5) were crossed with transgenic mice expressing the cognate antigenic protein under the control of the H- 2K^b promoter. Double-transgenic mice show negative selection of thymocytes at the CD4⁺8⁺TCR¹⁰ to CD4⁺8⁺TCR^hi transition stage. A few CD8 T cells, however, escape clonal deletion, and in the peripheral lymphoid organs of these mice, they exhibit low levels of the transgenic receptor and upregulated levels of the CD44 memory marker. Such cells do not proliferate upon exposure to antigen stimulation in vivo or ex vivo, however, they can develop low but detectable levels of antigen-specific cytotoxic function after stimulation in vitro in the presence of IL-2.     

Predominant and stable T cell responses to regions of myelin basic protein can be detected in individual patients with multiple sclerosis

T lymphocytes from patients with multiple sclerosis (MS) recognize multiple myelin basic protein (MBP) epitopes. This situation complicates the design of specific immunotherapies. We investigated to which extent the T cell response to MBP is heterogeneous in single subjects in terms of preferentially recognized regions of the molecule, major histocompatibility complex (MHC) restriction, and stability over time. From each of nine patients with MS, a minimum of six MBP-specific T lymphocyte lines (TLL) were assayed for the proliferative response to a panel of overlapping peptides, encompassing the whole MBP. Predominant Tcell recognitions of distinct MBP regions were present in three patients, all HLA-DR2⁺, independently of the clinical features of their disease. Tcell reactivity was preferentially directed to residues 16-38 in one patient. In this case the response was also stable over time, during different phases of the disease. Predominant reactivity to residues 86-99 was detected in the two other DR2⁺ patients. In each of the patients with other HLA-DR haplotypes (DR2^?), as well as in three DR2⁺ non-MS donors, the Tcell response to MBP appeared to be considerably more heterogeneous. The HLA restriction element varied among TLL recognizing the same MBP region, even when raised from the same individual. The genomic HLA typing, performed on the DRB1 and DRB5 genes in the DR2⁺ subjects, showed no obvious correspondence between preferential responses to regions of MBP and HLA-DR2 subtypes. In this context, a simple, new method for the genomic typing of the HLA-DRB1 gene in individuals with the HLA-DR2 serological specificity is also described. We conclude that predominant and stable T cell responses to a single MBP region can be detected in some patients with MS. In these individuals, the MHC restriction of the T cell recognition of predominant regions appears to be variable. Polymorphisms of the HLA-DR2 gene products alone do not account for the selection of the dominant MBP Tcell epitope.     

The non-immunosuppressive immunophilin ligand GPI-1046 potently stimulates regenerating axon growth from adult mouse dorsal root ganglia cultured in Matrigel

We used explant cultures of adult mouse dorsal root ganglia with spinal nerve attached growing in Matrigel to assess the effects of the non-immunosuppressive immunophilin ligand GPI-1046 [Snyder et al. (1998) TIPS 19, 21-26] on the growth rate of regenerating sensory axons and found a potent stimulation of axon growth. In these explant cultures, naked, unfasciculated axons emerge from the cut end of the spinal nerve and continue to grow in the Matrigel for up to eight days [Tonge et al. (1996) Neuroscience 73, 541-551]. Some axons are entirely smooth whilst others show prominent varicosities. Some of the former express the phosphorylated neurofilament epitope recognised by monoclonal antibody RT97, a marker for large calibre, myelinated axons, whilst the latter express calcitonin gene-related peptide, predominantly a marker for unmyelinated, and small diameter myelinated sensory axons. Many of the axons in these cultures also express the low-affinity neurotrophin receptor p75. GPI-1046 has been shown to have striking stimulatory effects on embryonic primary sensory axons growing in vitro and it was therefore of interest to see whether it could also enhance regenerating sensory axon growth from the adult ganglia in our cultures. GPI-1046 potently stimulated axon growth in our cultures in a dose-dependent manner. The stimulatory effect was not dependent on the class of sensory axon. These observations show that GPI-1046 is a potent stimulator of regenerating axons from adult, primary sensory neurones. The cellular site of action of GPI-1046 is unknown. To distinguish between a direct effect of the drug on neurones and an indirect effect we compared the effects of GPI-1046 on explant and dissociated cultures. In confirmation of previous results, we found that GPI-1046 potently stimulated axon outgrowth from explants of embryonic chick dorsal root ganglia. However, the drug was without effect on dissociated embryonic dorsal root ganglion neurones, suggesting that non-neuronal cells are important for axon growth stimulation.     

Study of folate receptor genes in nonsyndromic familial and sporadic cleft lip with or without cleft palate cases

Folate receptor family members (FOLRs) mediate the delivery of 5-methyltetrahydrofolate to the interior of, out of within, or between cells in a process known as potocytosis. Three FOLRs and a pseudogene map to 11q13.4. The aim of this study was to verify whether FOLRs could be responsible for the onset of nonsyndromic cleft lip with or without cleft palate (CL/P). Linkage and linkage disequilibrium between genetic markers and disorder were analyzed. Patients and their mothers from 71 familial CL/P pedigrees and 75 sporadic cases from Italian population were investigated by PCR-SSCP analysis. Data from mutation scanning allowed us to find only a silent mutation in FOLR1 present in a mother and her child. Our findings do not support FOLR1 and FOLR2 genes in the onset of CL/P.     

Meiotic recombination in the beta globin gene cluster causing an error in prenatal diagnosis of beta thalassaemia.

In the course of a prenatal diagnosis for beta thalassaemia by linkage analysis of restriction fragment length polymorphisms, a homozygous beta thalassaemia fetus was misdiagnosed as beta thalassaemia trait. Extensive studies of the polymorphic sites within the beta globin gene cluster in all the members of the family resulted in the conclusion that the paternal chromosome 11 of the newborn was different from that expected. Paternity was confirmed by HLA typing and blood group studies. The analysis of another polymorphic locus on chromosome 11 within the family was in agreement with the possibility of a crossing over between the two paternal chromosomes in a region 5' to the beta gene, previously indicated to contain a 'hot spot' area for recombination. This report underlines the risk of performing prenatal diagnosis using restriction polymorphisms 5' to the beta gene.     

Molecular cytogenetic characterization of a complex rearrangement involving chromosomes 9 and 22 in a case of Ph-negative chronic myeloid leukemia

The "golden path", produced by the Human Genome Project effort, is composed of a collection of overlapping and fully sequenced BAC/PAC clones covering almost completely the human genome. These clones can be advantageously exploited as fluorescence in situ hybridization (FISH) probes for the characterization of rearrangements frequently found in tumors. Breakpoint characterization can be further refined by generating additional smaller FISH probes through LONG-PCR amplification of specific DNA segments, 5-10 kb in size, using appropriate BAC/PAC probes as template. We report here an example of this approach that has been used to characterize a complex Ph-negative chronic myeloid leukemia (CML Ph-) case in which the BCR/ABL fusion gene was found located on chromosome 9.     

Development of an ELISA test for determination of the urinary trypsin inhibitor: analytical performance and applications

Increased urinary excretion of urinary trypsin inhibitor (UTI) has been reported in various inflammatory conditions and in Alzheimer's subjects, but its diagnostic potential remains to be elucidated. A reliable and specific enzyme-linked immunosorbent assay (ELISA) test for the determination of the UTI in human urine was developed. This assay was performed using 96-well microtiter plates. The plate surface is coated with an anti-UTI polyclonal antibody, the urine sample was added in a dilution range, and the detection was achieved using the enzyme-conjugated antibody. The assay was quantified by the build-up of colored product upon the addition of the substrate. Recoveries were 93%, and the intra- and inter-assay CVs were 4.25% and 21%, respectively. The ELISA showed parallelism of standard and urine samples and no significant interference by the biological matrix. The usefulness of the assay has been demonstrated by applying it to urine samples from Alzheimer's disease patients, and comparing with negative controls. UTI urinary levels are significantly increased in Alzheimer's subjects.     

Interactions between taste receptors in the frog tongue

Summary Receptors sensitive to stimulation with dilute CaCl₂ solutions and located in different fungiform papillae of the frog tongue are cross-connected via branches of the glossopharyngeal nerve fibers to form chemical sensory units comprising on average about 3 papillae; often one or more papillae were found to be common to different units.The receptor response observed when single papillae were individually stimulated increased, when the strength of the stimulus was raised, at a much lower rate than that obtained by stimulating the whole tongue surface. This proves that the antidromic impulses travelling in the cross-connections linking different papillae result in an evident depression of the receptor response to CaCl₂.The possible functional significance of mutual interaction between the receptors in the frog tongue is discussed.This study was supported by grants from the Consiglio Nazionale delle Ricerche (Impresa di Elettrofisiologia) of Rome, Italy.