Increased chromosomal instability in lymphocytes from elderly humans

Lymphocytes from young and old donors were incubated with PHA for 96 h and exposed to [3H]Tdr during the last 24 h of culture. Comparable amounts of [3H]Tdr were incorporated into chromosomes of old and young lymphocytes as measured by autoradiography of metaphase chromosomes. However, chromosomal damage and cell-cycle arrest were far greater in lymphocytes from old as compared to young humans. The frequency of chromosome breaks, fragments, exchange figures and dicentric chromosomes induced by [3H]Tdr was greater in cultures from old than in cultures from young humans. Lymphocytes from old donors exposed to 20 microM BrdU during the last 24 h of culture showed significantly more sister-chromatid exchanges than did lymphocytes from young donors. These data suggest that chromosomes in lymphocytes from old donors express more damage after exposure to [3H]Tdr or BrdU than do chromosomes in lymphocytes from young donors.     

"Cross-wiring" of the immune response in old mice: increased autoantibody response despite reduced antibody response to nominal antigen

Older humans and experimental animals have been repeatedly found to have higher titers of autoantibodies than do younger individuals despite the impaired responses of older individuals to foreign antigens. The studies reported here were designed to examine the relationship between these two age-related changes in antibody responses. Antibody response to foreign antigen was measured concurrently with autoantibody response in the same mice. Old mice (18-24 months old) had decreased responses to foreign antigens and increased responses to bromelain-treated syngeneic erythrocytes, compared to young mice (2 months old). In vitro mixing experiments were consistent with the possibility that suppressor cell activity in spleen cells from old mice reduce the antibody response to foreign antigen but not to autologous antigen. The results support an emerging view that age-associated changes in immune responses are the result of dysregulation rather than exhaustion of the immune system.     

Effect of age on ease of B-cell tolerance induction

It was shown in intact mice, and in lethally irradiated, thymectomized recipients of peripheral B cells from donors of various ages, that the ease of B-cell tolerance induction with the dinitrophenyl derivative of the copolymer of d-glutamic acid and d-lysine decreases with age. That is, the B-cell population of 6- to 7-month-old BALB/c mice is more resistant to tolerance induction than is the B-cell population of $\text{1}\text{1}\text{2}$- to 2-month-old mice and the B-cell population of 10- to 11-month-old mice is highly resistant to tolerance induction. This age-related resistance of the peripheral B-cell population to tolerance induction might, at least in part, account for the increased incidence of autoantibodies in aged animals. However, the increased resistance to tolerance induction appears to occur at an earlier age than the increased incidence of autoantibodies. This suggests that changes in ease of tolerance induction may not be the sole factor responsible for the increase in autoantibodies in aged animals. Evidence is also presented for an age-related decrease in the function of the B-cell population with respect to the response to a T-dependent antigen.     

Regulation of immune response in allogeneic mixed spleen cell cultures. I. Influence of I-region on the generation of suppressor cells

The immune responses of allogeneic mixed spleen cell cultures (MLC) to the T-dependent antigen, SRBC, and to the T-independent antigen, DNP-PAA, were investigated. The immune response to DNP-PAA in MLC with certain strain combinations was always suppressed as compared with the expected PFC response calculated from the PFC responses of the individual strains. This suppression was eliminated by treating the spleen cells with RAMB antiserum plus complement before the incubation of the MLC with DNP-PAA. It can be concluded that the suppression in the PFC response to the T-independent antigen DNP-PAA in MLC is due to the generation of suppressor T-cells. The PFC response to the T-dependent antigen, SRBC, in MLC showed either suppression, no change, or rarely augmenation, suggesting that the allogeneic mixed spleen cell cultures can generate both suppressor and helper T cells and that the balance between helper and suppressor activity regulates the PFC response to a T-dependent antigen. Suppressor activity was also generated in a one-way MLC, but the degree of suppression depended upon which of the two strains was responding. Similar amounts of thymidine were incorporated in the one-way MLR irrespective of which strains was responding. Thus, the extent of proliferation in one-way MLR is not related to the degree of suppressor activity generated. The results further indicate that a difference between two strains in the I-C, S, and G regions of the major histocompatibility complex is required to generate suppressor activitiy that can depress the response to a T-independent antigen, MLC between strains differing in K, I-A, I-B, I-J, I-E, and D regions generate little or no suppressor activity in this system.     

Immunological studies of aging. IX. Impaired proliferation of T lymphocytes detected in elderly humans by flow cytometry

Lymphocytes from humans over the age of 65 incorporate approximately 50% less tritiated thymidine than do lymphocytes from young donors when cultured with phytohemagglutinin. Because lymphocytes from elderly humans are more sensitive to cell cycle arrest induced by tritiated thymidine, it was impossible to determine to what extent impaired thymidine incorporation reflected a defect in proliferation or the increased sensitivity to the radioactive isotope. Flow cytometry was used to measure the proliferative response of T cells from young and old donors in culture with PHA. It was found that 25 percent fewer lymphocytes from old as compared to young humans enter the G1 or complete the S phase of the cell cycle. However, the rate of progression through the cell cycle by activated cells from young and old humans is comparable. Thus, flow cytometry suggested that the difference in thymidine incorporation by lymphocytes from old and young donors is attributable equally to a proliferative defect and to cell cycle arrest induced by tritiated thymidine. This conclusion was supported by the fact that the relative impairment of thymidine incorporation by lymphocytes from old donors was only one-half as great when a 20-min instead of a 24-hr pulse of tritiated thymidine was used.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Stimulation of endothelial IL-8 (eIL-8) production and apoptosis by phenolic metabolites of benzene in HL-60 cells and human bone marrow endothelial cells

Benzene toxicity is considered to be elicited by its metabolites and phenolic metabolites of benzene are known to induce apoptosis in leukemia cells in culture and in human bone marrow progenitor cells. One potential mechanism of apoptosis induced by benzene metabolites that has not been examined is the production of pro-apoptotic cytokines such as endothelial IL-8 from endothelial cells in bone marrow stroma. In this study, we utilized HL-60 cells which are known to produce the endothelial form of IL-8 (eIL-8) and human bone marrow endothelial cells (HBMEC) as model systems. Hydroquinone (HQ), Catechol (Cat) and benzenetriol (BT) all induced eIL-8 production and apoptosis in HL-60 cells. HQ induced a marked 50-70 fold stimulation of eIL-8 levels and HL-60 cells were shown to have the eIL-8 receptor, CXCR1 thus enabling an autocrine pathway of apoptosis. However, treatment with recombinant eIL-8 failed to induce apoptosis in HL-60 cells as previously reported and antibodies to either IL-8 or CXCR1 did not significantly abrogate benzene metabolite-induced apoptosis. HQ and Cat but not BT also induced stimulation of eIL-8 production in HBMEC. These data demonstrate that although metabolites of benzene induce marked stimulation of eIL-8, this is unlikely to be responsible for apoptosis induced in HL-60 cells. Our data also demonstrates that phenolic metabolites of benzene stimulate the production of eIL-8 from HBMEC suggesting that higher levels of endothelial-derived cytokines may occur in bone marrow after benzene exposure.     

Stimulation of endothelial IL-8 (eIL-8) production and apoptosis by phenolic metabolites of benzene in HL-60 cells and human bone marrow endothelial cells

Benzene toxicity is considered to be elicited by its metabolites and phenolic metabolites of benzene are known to induce apoptosis in leukemia cells in culture and in human bone marrow progenitor cells. One potential mechanism of apoptosis induced by benzene metabolites that has not been examined is the production of pro-apoptotic cytokines such as endothelial IL-8 from endothelial cells in bone marrow stroma. In this study, we utilized HL-60 cells which are known to produce the endothelial form of IL-8 (elL-8) and human bone marrow endothelial cells (HBMEC) as model systems. Hydroquinone (HQ), Catechol (Cat) and benzenetriol (BT) all induced eIL-8 production and apoptosis in HL-60 cells. HQ induced a marked 50-70-fold stimulation of eIL-8 levels and HL-60 cells were shown to have the eIL-8 receptor, CXCR I thus enabling an autocrine pathway of apoptosis. However, treatment with recombinant elL-8 failed to induce apoptosis in HL-60 cells as previously reported and antibodies to either IL-8 or CXCRI did not significantly abrogate benzene metabolite-induced apoptosis. HQ and Cat but not BT also induced stimulation of elL-8 production in HBMEC. These data demonstrate that although metabolites of benzene induce marked stimulation of eIL-8, this is unlikely to be responsible for apoptosis induced in HL-60 cells. Our data also demonstrates that phenolic metabolites of benzene stimulate the production of eIL-8 from HBMEC suggesting that higher levels of endothelial-derived cytokines may occur in bone marrow after benzene exposure.