Hepatitis B immunization of healthy elderly adults: relationship between naïve CD4+ T cells and primary immune response and evaluation of GM-CSF as an adjuvant.

The efficacy of granulocyte-macrophage colony-stimulating factor (GM-CSF) to enhance the primary immune response to hepatitis B vaccine was studied in healthy elderly with young volunteers included as controls in this double-blind, placebo-controlled trial of GM-CSF as an immune adjuvant. Naïve T-helper cells (CD4+CD45RA+) were determined at baseline. Forty-five healthy elderly (average age, 74 years) and 37 healthy young controls (average age, 28 years) were randomized. Hepatitis B vaccine was administered at 0, 1, and 6 months. GM-CSF as a single injection of either 80 microg or 250 microg with the first and second doses of hepatitis B vaccine. In this trial GM-CSF did not enhance antibody responses. However, the antibody responses were dramatically different between these two groups: 35/35 young developed a protective titer versus 19/45 elderly (P < 0.0001). In addition, the mean logarithm of anti-hepatitis B antibody level in the 35 young who completed the study was 3.17 (log mIU/ml) but only 2.21 in the 19 elderly responders (P < 0.0001). Naïve T-helper cells differed significantly between the two groups: the mean percentage of CD4+CD45RA+ T cells was 47.9% versus 35.0% (P < 0.0001) in the young and elderly volunteers respectively. Naïve T cells also differed significantly between elderly who did or did not respond to HBV (39.9% vs. 31.7%, P = 0.039). Using linear regression, age, and percent naive, CD4 T cells were determined to significantly influence the anti-hepatitis B antibody response, but sex and dose of GM-CSF did not. For a two-parameter model: logarithm of antibody titer = (-0.038 x age in years) + (0.031 x % naïve CD4T cells) + 2.68; adjusted r2 = 0.605 and P < 0.0001. However, age had a larger effect than naive CD4 T cells, i.e., in comparing young and elderly groups the log antibody titer decreased by 1.73 due to the increase in age but only 0.40 due to the decrease in naive CD4 T cells. Thus, there was a large effect of age that could not be explained by the quantitative change in the naïve T-helper cells.

Response of C-reactive protein and serum amyloid A to influenza A infection in older adults.

Influenza epidemics are associated with significant morbidity and mortality in the elderly, with a substantial proportion of deaths due to cardiovascular events. Elevations of acute-phase proteins have been associated with an increased risk of atherosclerotic events. Therefore, serum amyloid A (SAA) and C-reactive protein (CRP) were measured during influenza illness and 4 weeks later in 7 young persons, 15 elderly outpatients, and 36 hospitalized adults. Striking elevations were seen in mean acute SAA and CRP levels in all groups, but hospitalized patients had the highest levels (SAA, 503 vs. 310 microg/mL [P=.006]; CRP, 120 vs. 34 microg/mL [P<.001]). The presence of dyspnea, wheezing, and fever was also associated with high CRP levels. Influenza infection is associated with significant elevations of SAA and CRP levels in elderly patients, especially those who require hospitalization. It is possible that direct effects of CRP may exacerbate preexisting atherosclerotic lesions and may help explain cardiovascular events associated with acute influenza.

Aging and T-cell-mediated immunity.

Changes in the T-lymphocyte compartment represent the most critical component of immunological aging. Recent studies have demonstrated that the age-related decline in T-cell-mediated immunity is a multifactorial phenomenon affecting T-cell subset composition as well as several proximal events such as protein tyrosine phosphorylation, generation of second messengers, calcium mobilization and translocation of protein kinase C, and distal events such as lymphocyte proliferation and cytokine production of the T-cell activation pathway. Age-related T-cell immune deficiency is preceded by thymic involution and is influenced by several intrinsic as well as extrinsic factors. Further, the role of monocytes and macrophages in T-cell activation changes with advancing age. This brief review will summarize the current knowledge of the cellular as well as molecular aspects of immunodeficiency of T cells due to aging, some of the paradoxes of aging as related to T-cell-mediated immunity, and possible factors which contribute to this paradox. Finally, experimental approaches will be suggested that might resolve these controversies and that might provide insights into the diverse and complex mechanisms that contribute to immunodeficiency of T cells. Ultimately these studies may suggest possible therapeutic interventions to enhance immune function in the elderly.

Role of cytomegalovirus in the T cell changes seen in elderly individuals.

The effect of prior cytomegalovirus (CMV) infection on the immune system was evaluated in young and elderly volunteers. Prevalence of IgG antibodies to CMV was higher in the elderly volunteers. In both age groups, there was a strong association with CMV seropositivity and increased number of CD28- CD4 or CD8 T cells, as well as with increased numbers of T cells expressing CD56 or DR. Although these changes have previously been reported to be age-related, they were independent of age when CMV serological status was taken into account. In contrast, both age group and CMV status were important determinants of the total number of T cells, the number of CD8 T cells, and the number of CD8 T cells expressing CD45RA or CD28. These findings indicate that prior infection with CMV, as reflected by CMV serological status, has important effects on T cell subsets and surface markers and must be considered whenever evaluating age-related changes in immunological parameters.

Effect of age on mitogen induced protein tyrosine phosphorylation in human T cell and its subsets: down-regulation of tyrosine phosphorylation of ZAP-70.

Several events of T cell activation have been reported to decline in humans with age. Since protein tyrosine phosphorylation is an early critical event of T cell activation, we performed a systematic analysis of the age-associated changes in the mitogen induced protein tyrosine phosphorylation of human T lymphocytes using SDS-PAGE and Western blotting techniques. Following stimulation with Con A and PHA, an identical pattern of protein tyrosine phosphorylation was observed in the lysates of T cells prepared from seven healthy young adults and eight healthy elderly human subjects. Five different high molecular mass proteins (75, 115, 120, 140 and 170 kDa) were consistently tyrosine phosphorylated in all of the donors from both age groups and peaked between 3 and 10 min. Tyrosine phosphorylation of the above substrates was observed in both CD4 and CD8 subsets. When compared for individual donors from both age groups, variations in the T cell response with regard to net tyrosine phosphorylation for all the substrates was observed. However, the mitogen induced level of tyrosine phosphorylation of only p75 was found to be significantly lower in unfractionated T cells as well as CD4 and CD8 subsets of older subjects than that of young subjects. Using immunoblotting, p75 was identified as ZAP-70, a member of the syk family of protein tyrosine kinases. Understanding of the biochemical basis of the reduced level of tyrosine phosphorylation of ZAP-70 will be helpful in delineating the molecular basis of age-associated impairment of T cell activation.

The MoAb-V kappa IIIb cross-reactive idiotope on A27a (Humkv325) encoded kappa chains maps to framework region 3. off.

The monoclonal antibody MoAb-V kappa IIIb binds a cross-reactive idiotopic (CRI) determinant on light (L) chains encoded by the V kappa IIIb subgroup A27a (Humkv325) gene segment. The aim of this study was to localize the MoAb-V kappa IIIb CRI. Mutational analyses involving region exchanges between a CRI-positive V kappa IIIb chain and a CRI-negative V kappa 1 chain indicate that the MoAb-V kappa IIIb CRI is located in framework region (FR) 3 of A27a (Humkv325) encoded L chains. CRI-positive kappa chains unpaired with a heavy (H) chain are reactive with MoAb-V kappa IIIb, indicating that the CRI is located on the kappa chain alone without involvement of H chain residues. Combinatorial antibodies composed of non-parental L and H chain pairings are reactive with MoAb-V kappa IIIb only when the L chain is A27a (Humkv325) encoded. The CRI, therefore, is not readily perturbed by H chain interactions. When the FR3 from a CRI-positive kappa chain replaced the FR3 in a CRI-negative lambda chain, the determinant was no longer detectable with MoAb-V kappa IIIb. It is possible, therefore, to exchange regions between kappa chains from different families and retain the CRI structure, however the determinant is lost when placed in a more foreign background such as a lambda chain. These data more precisely define the interaction between MoAb-V kappa IIIb and its CRI, and indicate that there are limits within which antibody FRs can be shuffled and still retain their native structural features.

Human IgG production in vivo: determination of synthetic rate by nonradioactive tracer incorporation.

Exposure of Ab-secreting cells to selected hormones, cytokines, and drugs alters the rate of Ig production in vitro. Whether these effects are important clinically is unknown because there are no safe, reproducible, and appropriate techniques for measuring Ig synthesis in vivo. We developed a stable isotope tracer method to measure IgG secretion into plasma. L-[1-13C]Leucine was given as a priming dose followed by a constant i.v. infusion over 8 h. Tracer accrual in IgG, determined by mass spectrometry, was linear from 2 to 8 h of the infusion. The normal rate of IgG synthesis into plasma assessed in 21 healthy subjects was 860 +/- 310 mg/day (mean +/- SD). The synthetic rate measurements were remarkably reproducible (coefficient of variation = 10.5 %). Simultaneous analysis of leucine kinetics allows, for the first time, Ig secretion to be studied in the context of whole body protein economy. IgG secretion into plasma accounts for 0.3% of whole body protein synthesis. Experimental support for a key metabolic assumption, that tracer enrichment in plasma and that at the site of IgG synthesis were similar, came from a comparison of synthetic rates derived from low dose and high dose tracer infusions. Measurement of Ig secretion by tracer incorporation is rapid and reproducible. In contrast to older methods that rely on radioisotope disappearance, the tracer incorporation method is safe for serial measurements in an individual and will be useful for quantitative studies of treatment effects and immune regulation in vivo.

Gene-modified cells for the treatment of cancer.

Gene therapy involves the insertion of a gene into an organism to treat a disease. Since its early development in the 1970s, gene therapy has expanded rapidly both in terms of the methods available and the number of candidate diseases for treatment. This report reviews gene therapy for cancer, including methodology, pre-clinical studies and experimental clinical trials.

Apoptosis and interleukin 7 gene expression in chronic B-lymphocytic leukemia cells.

mRNA for interleukin 7 (IL-7) was readily detected in leukemic cells immediately upon their removal from patients with chronic B-lymphocytic leukemia (B-CLL). IL-7 mRNA expression and IL-7 gene transcription were down regulated, however, when B-CLL cells were placed in culture at 37 degrees C for 4 hr. Down regulation of the IL-7 gene was prevented in cells maintained at 4 degrees C. Continued culture of B-CLL cells at 37 degrees C resulted in programmed cell death, or apoptosis, as evidenced by DNA fragmentation. The coincident kinetics of IL-7 gene down regulation and apoptosis suggested that IL-7 gene expression may be required for maintenance of CLL viability in vivo. Signals for IL-7 gene regulation and apoptosis induction were thus examined. Activation of normal B cells through their immunoglobulin receptors did not result in upregulation of IL-7 gene expression. Reagents required for CLL cell purification and culture also did not contribute to IL-7 gene regulation and apoptosis induction. IL-7 gene expression was retained and apoptosis was prevented, however, in CLL cells cultured on a monolayer of EA.hy926 human umbilical cord endothelial hybrid cells. Signals specifically presented by EA.hy926 cells supported both CLL cell viability and IL-7 gene expression, whereas culture of CLL cells on A549/8 carcinoma cells, the fusion partner used to generate the EA.hy926 cells, did not. Cell-cell contact was required, as culture supernatants did not prevent apoptosis. Specifically, IL-7 mRNA expression was retained and apoptosis was prevented only by contact with the endothelial cell hybrids. Preliminary data indicated that integrins expressed on CLL cells affected modulation of apoptosis and IL-7 gene regulation, suggesting that integrins may play significant roles in regulating viability of CLL cells.

The "bystander effect": tumor regression when a fraction of the tumor mass is genetically modified.

Tumor cells expressing the herpes simplex virus thymidine kinase (HSV-TK) gene are sensitive to the drug ganciclovir (GCV). We demonstrate here that HSV-TK-positive cells exposed to GCV were lethal to HSV-TK-negative cells as a result of a "bystander effect." HSV-TK-negative cells were killed in vitro when the population of cultured cells contained only 10% HSV-TK-positive cells. The mechanism of this "bystander effect" on HSV-TK-negative cells appeared to be related to the process of apoptotic cell death when HSV-TK-positive cells were exposed to GCV. Flow cytometric and electron microscopic analyses suggested that apoptotic vesicles generated from the dying gene-modified cells were phagocytized by nearby, unmodified tumor cells. Prevention of apoptotic vesicle transfer prevented the bystander effect. The toxic effect of HSV-TK-positive cells on HSV-TK-negative cells was reproduced in an in vivo model. A mixed population of tumor cells consisting of HSV-TK-positive and HSV-TK-negative cells was inoculated s.c. into mice. Regression of the tumor mass occurred when the inoculum consisted of as few as 10% HSV-TK-expressing tumor cells. The bystander effect was also demonstrated in i.p. tumor studies. Initial experiments demonstrated that prolonged survival (> 70 days) occurred when a mixture containing 50% HSV-TK-positive and 50% HSV-TK-negative cells was injected i.p. followed by GCV treatment. Further, survival was prolonged for mice with a preexisting HSV-TK-negative i.p. tumor burden by injecting HSV-TK-positive cells and GCV. These results suggest that genetic modification of tumor cells may be useful for developing cancer therapies.

Oncogene elements within an endogenous retrovirus.

The human genome contains a large number of endogenous retroviral-related sequences. While the function of these sequences is unknown, they may contribute to disease processes through their regions of homology with infectious retroviruses. We have been further characterizing a recently reported HTLV-1 related endogenous retroviral sequence cloned from T lymphocytes isolated from a patient with essential cryoglobulinemia. We here report further detailed transcriptional analysis of the sequence for tissue and cell-cycle specificity and a novel finding of an association between the endogenous retrovirus and a ras-related gene.

Antibodies to retroviral proteins and reverse transcriptase activity in patients with essential cryoglobulinemia.

Human T cell lymphotropic virus type I (HTLV-I) gag and env protein-specific antibodies were identified in 6 of 13 patients with essential cryoglobulinemia (EC), by Western blot and radioimmunoprecipitation analysis. Supernatants of cells from 2 of the 5 EC patients tested showed reverse transcriptase activity. DNA sequences homologous to HTLV-I could not be detected by polymerase chain reaction, thus excluding the presence of prototype HTLV-I in each patient with EC. The data suggest that retroviral proteins distinct from but related to HTLV-I may be involved in the pathogenesis of EC in some patients.

Estimation of serum V kappa IIIb light chains in rheumatoid arthritis and correlation with CD5-positive B-cells.

The presence of V kappa IIIb light chains in the sera of rheumatoid arthritis (RA) patients has been evaluated by an enzyme-linked immunosorbent assay (ELISA). V kappa IIIb light chains have been confirmed to be largely restricted to IgM, and were rarely detected in the IgG fraction of sera. The concentration of total serum V kappa IIIb did not significantly vary with age, nor did it correlate with IgM-rheumatoid factor (RF) titre. Although total serum V kappa IIIb was not significantly increased in RA patients compared with matched controls, IgM-RFs frequently contained V kappa IIIb. Using flow cytometry, CD5-positive B-cells were not increased in these RA patients compared with healthy laboratory control personnel. Furthermore, there was no direct correlation between total serum IgM V kappa IIIb content and CD5-positive B-cell numbers in peripheral blood.

[Clonal immunoglobulin rearrangement in patients with essential cryoglobulinemia]. / Klonális típusú génátrendezödések "essentialis" cryoglobulinaemiás betegekben.

'Essential' cryoglobulinemias (EC) are characterized by the precipitation of immunoglobulins (Igs) from the sera of patients at temperatures below 25 degrees C. The cryoprecipitate of type I EC patients is comprised of a monoclonal Ig while that of patients with type II or 'mixed' EC contains monoclonal auto-antibodies with rheumatoid factor activity. In order to define if the high cryoglobulin production rate is related to a clonal B-cell expansion the rearrangement of Ig genes was investigated by Southern blot analysis of DNA extracted from peripheral blood lymphocytes of EC patients. Clonal expansion of B cells could be detected using Ig light and heavy chain specific DNA probes in 3/4 patients with type I EC and 4/12 patients with type II EC. In the group of patients with clonal Ig gene rearrangements, in two cases with type I EC and one case with type II EC, alterations of the c-myc locus was also noted. Demonstration of clonal B-cell expansions in EC patients shows that the clonal type of Ig gene rearrangements is not a unique marker of malignant lymphomas. Since malignant B-cell lymphomas can develop in a small number of EC cases, the follow-up of these patients should be pursued indefinitely.

Rearrangement of the T-cell receptor alpha, beta and gamma chain genes in chronic lymphocytic leukemia.

Simultaneous expression of mature B-cell and T-cell markers and subsequent abrogation of expression of the T-cell surface markers by cytotoxic chemotherapy was reported earlier in a patient (TG) with chronic lymphocytic leukemia (CLL). In addition to rearrangements of the immunoglobulin (Ig) gene loci correlating with phenotypic data, the T-cell receptor (TCR) alpha, beta and gamma chain genes also displayed clonal rearrangements in peripheral blood lymphocyte DNA of TG. The present case shows that in CLL cells not only the expression of B-cell and T-cell specific differentiation antigens but also the rearrangement of Ig as well as TCR alpha, beta and gamma genes may occur simultaneously.

Detection and cloning of new HTLV-related endogenous sequences in man.

Human T-cell leukemia virus (HTLV) type I-related endogenous sequences (HRES) have been cloned from a human genomic library. HRES-1/1 is present in DNA of all normal donors examined. By nucleotide sequence analysis, HRES-1/1 contains two potential open reading frames capable of encoding a p25 and a p15. A 684 bp flanking region 5' from the first ATG codon of p25 contains a TATA-box, a poly-adenylation signal, a putative tRNA primer binding site, and inverted repeats at locations which are typical of a retroviral long terminal repeat. Phylogenetic analysis suggests that HRES-1/1 entered the genome in primates, presumably as an exogenous retrovirus. From the deduced amino acid sequence of HRES-1/1 p25, residues 6-36 show a sequence homology of 32% and 39% to gag region segments of HTLV-I and HTLV-II, while residues 104-139 display a sequence homology of 33% and 28% to the gag regions of human immunodeficiency virus type 2 (HIV-2) and feline sarcoma virus (FSV), respectively. This suggests that the original exogenous virus infecting primate may be chimeric in structure. The HRES-1/1 genomic locus is transcriptionally active in lymphoid cells, melanoma cells, and embryonic tissues.

Detection and mapping of polymorphic KpnI alleles in the human T-cell receptor constant beta-2 locus.

Southern blot analysis with human T-cell receptor (TcR) beta-chain specific cDNA probes revealed two novel allelic forms of the TcR beta-2 gene locus. Three different genotypes were noted based on the presence of polymorphic KpnI restriction fragments: I, 5.7 kb fragment only; II, 3.9 kb and 1.8 kb fragments only; III, all three polymorphic fragments. This hybridization pattern suggested that the presence or absence of a polymorphic KpnI site within the 5.7 kb fragment defines the two different allelic forms of the TcR beta chain locus. By Southern blot analysis of genomic DNA from T-cell lines with deleted C-beta-1 regions and computer-assisted restriction site mapping of germline and cDNA sequences of the C-beta-2 locus, the polymorphic KpnI site was localized at 24 bp 5' to the third exon of the C-beta-2 gene. It was determined that the polymorphic KpnI site and the earlier described polymorphic BglII site located 5' to the C-beta-2 gene are not co-inherited. No difference was noted in distribution of the KpnI genotypes and allelic frequencies between 26 normal individuals and 22 patients with systemic lupus erythematosus. However, this newly characterized polymorphism of the TcR locus should provide a useful tool to analyse the role of inherited genetic variations in the function of T lymphocytes under normal and pathological conditions.