Biomedical subjects
Publications and source records attributed to R J Cross.
After fullerenes are heated in the presence of a noble gas or an unreactive molecule at 650 degrees C and 3000 atm pressure, a small fraction of the fullerene molecules contain the atom or molecule. The incorporation fraction is greatly enhanced by adding potassium cyanide to the reaction mixture. The details of the preparation are described here.
3He NMR spectroscopy has been used to study the equilibria of Diels-Alder additions of 9,10-dimethyl anthracene (DMA) to (3)He@C(60) and (3)He@C(70). Spectra of a series of equilibrium mixtures showed peaks for the isomeric adducts. One monoadduct, six bis-adducts, eleven tris-adducts, and ten tetrakis-adducts of DMA to C(60) were seen. One monoadduct and three bis-adducts of C(70) were detected. Equilibrium constants were found for these reactions and values for DeltaG, DeltaH, and DeltaS were obtained.
In the rat lung, primary saccules are transformed into alveoli from postnatal Days 4 to 13, after which time there is a 20% reduction in the number of lung fibroblasts as the interstitial volume of the alveolar walls decreases. Our objective was to determine whether apoptosis is a factor in the observed decrease in the number of interstitial lung fibroblasts beyond Day 13. We used both histologic and flow cytometric assays to detect in lung fibroblasts the DNA fragmentation and condensation that are characteristic of apoptosis. In addition, we evaluated levels of bcl-2 and BAX messenger RNAs (mRNAs) using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Apoptotic cells were quantitated in glycol methacrylate-embedded sections of neonatal rat lungs using the terminal transferase dUTP-digoxygenin nick end-labeling (TUNEL) method. Although TUNEL-positive interstitial cells were observed in the lungs of rats ranging in age from 10 to 16 d, a dramatic increase in apoptotic cells was seen on Day 17. Although diminished in number, TUNEL-positive cells were still present on Day 28. Hoechst-stained apoptotic bodies were observed in isolated lung cells that were vimentin-positive and factor VIII-negative, which identified the apoptotic cells as fibroblasts as opposed to endothelial cells. Flow cytometric analysis of freshly isolated lung fibroblasts stained with Hoechst 33342 indicated a 24% increase in chromatin condensation in cells from 17-d versus 16-d rats. DNA fragmentation was also quantitated by flow cytometry in freshly isolated fibroblasts labeled with BODIPY-conjugated dUTP in the presence of terminal deoxynucleotidyl transferase. The percentage of lung fibroblasts containing fragmented DNA was 51.4 +/- 13.4 in 17-d, 36.9 +/- 8.6 in 18-d, and 13.8 +/- 5.4 in 19-d rat pups. Finally, evaluation by RT-PCR indicated that on postnatal Day 17, mRNA for bcl-2, which inhibits apoptosis, was decreased to 73.5 +/- 11.4% (P < 0.001) of Day 5 controls; whereas mRNA for BAX, which enhances apoptosis, was increased to 243.0 +/- 102.0% (P < 0.001) of Day 5 values. These results demonstrate that rat lung fibroblasts undergo apoptosis after the completion of alveolarization, and suggest that this decrease in fibroblast number plays an important role in the thinning and remodeling of the alveolar walls of the lung.
Insulin-like growth factor I (IGF-I) has been shown to be important in the maintenance, development, and proliferation of various types of leukocytes, particularly T cells. Radio-receptor binding assays demonstrate that Jurkat T cells bind 125I-IGF-I with an affinity of 1.77 nM (Kd) and express approximately 230 receptors/cell. Specificity studies show insulin also binds the IGF-I receptor with an affinity 20-fold lower than that of IGF-I. Interaction of IGF-I with its receptor on Jurkat T cells induces the phosphorylation of tyrosine kinase which is detectable by Western blotting. The 95,000 MW protein detected is equivalent to the molecular weight of the beta chain of the IGF-I receptor described in other types of cells. These studies characterize the binding of IGF-I to its receptor on Jurkat T cells, demonstrate that IGF-I binding induces tyrosine phosphorylation, and support the hypothesis that IGF-I is important in the induction of T cell activation.
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Peritoneal macrophages from two strains of mice, BALB/c and C3H/HeN, were analysed by flow cytometry for a respiratory burst associated with the phagocytosis of Ehrlichia risticii. Resident murine peritoneal macrophages failed to respond with a respiratory burst after phagocytosing E risticii. In contrast, mice previously infected with E risticii yielded peritoneal macrophages that did generate a respiratory burst on phagocytosing ehrlichiae.
The Snell-Bagg hypopituitary dwarf mouse has been shown to be deficient in growth hormone, thyroxine, and prolactin. There are reports indicating that in addition to these neuroendocrine abnormalities, development of immune competence is also severely impaired in these animals. However, other studies indicate that the immunologic potential of these mice does not differ from their heterozygous littermate controls. Our data show that dwarf mice weaned at 21 days of age and killed at that time, or 7 days later, have reduced numbers of cells in both the spleen and thymus and the mitogen responsiveness of these cells is impaired. However, if mice weaned on day 21 are analyzed at 32 days of age or the mice are weaned at day 30 and analyzed 7 days later the ability to respond to mitogenic stimulation does not differ from controls. Further experiments show that dwarf mice weaned at 30 days of age have a normal complement of V-beta TCR as evidenced by immunofluorescence analysis as well as a primary antibody response to SRBC equivalent to that observed in normal littermates. Immunofluorescence analysis of CD4 and CD8 expression on thymocytes obtained from dwarf mice shows a distinct pattern dependent on the time of weaning and time of analysis. Initial analysis of thymocytes from dwarf mice weaned and killed at 21 days of age do not differ from controls. However, cells from dwarf mice weaned on day 21 and killed on day 28 are markedly different with a loss of immature CD4+/CD8+ cells and a corresponding increase in CD4+ and CD8+ mature thymocytes. In contrast, the phenotype of thymocytes obtained from dwarf mice weaned at 30 days of age and killed on day 37 did not differ from normal littermates. Collectively these studies indicate that hypopituitary dwarf mice lag behind their heterozygous littermates with respect to development of immunocompetence but normal immune responsiveness does develop by 32 days of age when the mice are weaned on day 21.
We showed earlier that life spans of murine B lymphocytes could be estimated by measuring the functional reactivities of normal B cells upon transfer into x-linked immunodeficient (xid) mice, which do not respond to anti-mouse IgM (anti-mu) antibodies and thymic-independent type-2 (TI-2) antigens. Here the same approach was adopted, to evaluate the life spans of B-lymphocytes from aged mice. Spleen cells from normal young and aged mice were transferred into young or aged xid recipients and the decay kinetics were followed by measuring the proliferative response to anti-mu and PFC response to TNP-Ficoll, a prototype TI-2 antigen. The results indicated that anti-mu reactive B cells of both young and aged mice decayed with similar non-linear kinetics. About 50% of the donor cells decayed in 8-10 days, whereas, the remaining decayed at a slower rate and it appeared that the median life-expectancy of this latter population could be at least 3 weeks. Essentially, there was no apparent difference in the decay kinetics of anti-mu reactive B cells of young and aged mice. Unlike anti-mu reactive cells, TNP-Ficoll reactive B cells showed 2-3-fold enhancement in the PFC response during the first 2 weeks, and persisted at least until 5 weeks post transfer. This result indicated that TNP-Ficoll reactive B cells are long-lived. Further, it was found that the turn over rate of TNP-Ficoll reactive B cells was also very similar in young and aged mice. The environment of the aged mice also did not appear to have any effect since the survival profiles of anti-mu reactive B cells were the same in young or aged xid recipients. Altogether, these results suggest that aging does not significantly alter the life spans of mature B lymphocytes.
There is evidence to indicate that the neuroendocrine and immune systems can interact. Thus, neuroendocrine hormones can modulate a variety of immune functions and there have been attempts to manipulate the neuroendocrine system of aged animals to enhance immune function. We have previously shown that the transplantation of a syngeneic pituitary gland under the kidney capsule of young adult mice elevates serum prolactin and enhances immune responsiveness. In the present study pituitary glands were transplanted under the kidney capsule of 22-month-old mice to determine if this maneuver can enhance a number of immunologic parameters. The results demonstrate that aged animals bearing transplanted pituitary grafts for 10 days did not exhibit any enhancement in their primary antibody response to sheep red blood cells, splenic T or B-cell mitogen responsiveness or restoration of thymic architecture. When these immunologic assessments are performed on animals bearing pituitary grafts for 28 days, the IgM and IgG primary antibody responses and splenic T-cell responsiveness are enhanced but repopulation of the thymus still does not occur. Importantly, this enhancement does not restore immunocompetence to levels observed in young mice.
Experiments were conducted with rats and mice to evaluate the effect of the consumption of endophyte (Acremonium coenophialum) and associated toxin(s) infected tall fescue on humoral and cellular aspects of immune function. Treatment diets were: (1) rodent chow (RC) or (2) rodent chow mixed 1:1 (w/w) with endophyte infected (E+) or (3) non-infected (E-) tall fescue seed. Rats fed the E+ diet in experiment 1 (43 days) exhibited a lower (P less than 0.05) serum titer to sheep red blood cell (SRBC) immunization than those fed the E- diet (38.4 vs 131.3). The E+ rats also had lower (P less than 0.01) white cell counts than either RC or E- groups (5225 vs 8959 and 7491/mm3). Spleen cells from mice fed the E+ diet for 37 days exhibited a reduced (P less than 0.05) response to the mitogens Concanavalin A and lipopolysaccharide. Flow cytometric analysis revealed a significant (P less than 0.01) 42% increase in T suppressor cell numbers in spleens of mice fed the E+ vs RC diets.
The efficacy of killing small cell lung cancer (SCLC) cells at the G1, S, and G2-M phase of the cell-cycle by a new 111In-bleomycin complex (111In-BLMC) was investigated. SCLC cells (N417, H526, H209) were synchronized by double thymidine block and assessed by DNA content with flow cytometry, and the period for the maximal accumulation of cells in S, G1, or G2-M phase was determined. Cells in different cell cycle phases were exposed to 0.9% NaCl, BLM, or 111In-BLMC for 1 hour and observed for colony formation. The survival of H526 cells treated with 111In-BLMC was 71% (for enriched S phase), 46% (G1), and 31% (G2-M). For N417 cells, it was 25% (S), 20% (G1), and 8% (G2-M) for 111In-BLMC and 18% (S), 33% (G1), and 10% (G2-M) for BLM. These results indicated that SCLC cells in G2-M were most sensitive and those in S phase were least sensitive to 111In-BLMC; cells in G1 phase were the least sensitive to BLM.
Transplantation of a pituitary graft under the kidney capsule and the resulting elevation of serum prolactin enhances the primary humoral antibody response to sheep red blood cells. Enhancement of the response is not due to marked changes in the percentage of T-cells and their subsets, B-cells, or the number of nucleated spleen cells. Quantitation of serum prolactin levels correlates well with the proportion of enhancement as mice with two grafts and higher levels of prolactin have increased responsiveness compared to mice with one graft. Systemic administration of mouse prolactin at the time of immunization also enhances the humoral immune response; however, if prolactin treatment is delayed and given 24 h after immunization, no potentiation of the response occurs. Thus, prolactin is enhancing the immune response by affecting an early afferent event in the induction of the immune response.
We have previously shown that depletion of central nervous system (CNS) catecholamines by injecting the neurotoxin 6-hydroxydopamine (6-OHDA) into the cisterna magna of C57B1/6 mice markedly impairs the humoral immune response to sheep red blood cells. This work extends these observations by showing that 6-OHDA treatment also inhibits the humoral antibody response to the T-cell-dependent antigen trinitrophenyl-keyhole limpet hemocyanin, but does not affect the response to the T-independent antigen trinitrophenyl-lipopolysaccharide. This treatment also impairs humoral responsiveness at peripheral lymphoid sites in addition to inhibiting natural killer cell activity. However, 6-OHDA treatment in vivo does not affect in vitro mixed lymphocyte responsiveness, mitogen-induced lymphocyte activation or antigen presentation by macrophages.
This study extends our previous findings, which indicate that depletion of CNS catecholamines has a marked inhibitory effect on humoral immune responsiveness. These data show that depletion of CNS catecholamines by injection of 6-hydroxydopamine (6-OHDA) into the cisterna magna in conjunction with immunization enhances the activity of a population of splenic T-suppressor cells as evidenced by the transfer of these cells to normal recipients. Increased suppressor cell activity does not result solely from 6-OHDA treatment, but rather requires concomitant immunization. Further characterization shows that these suppressor cells are not antigen specific. Hypophysectomy abrogates the effects of 6-OHDA injection suggesting that catecholamine depletion modulates immune function via the release of pituitary hormones. Thus, depletion of CNS catecholamines impairs immune responsiveness by inducing enhanced T-suppressor cell activity, providing additional evidence of the involvement of the CNS in regulation of immune responsiveness.
Previous studies from this laboratory and others show that perturbations of the central nervous system modulate immune function. In addition, reports from several investigators indicate that depletion of the neurotransmitter norepinephrine (NE) in peripheral nerves by injecting the neurotoxin 6-hydroxydopamine (6-OHDA), can enhance or suppress the antibody response. However, immunocompetence following brain depletion of catecholamines has not been investigated. In this study, we investigated the effects of injecting 6-OHDA into the cisterna magna of male CBA/J mice, and determined the effects of this treatment on both the IgM and IgG antibody responses to sheep red blood cells (SRBC). Both responses are suppressed compared to saline-injected control or normal animals. Animals treated with 6-OHDA have decreased levels of NE in the midbrain, pons-medulla and hypothalamus, while dopamine levels did not change in these brain regions but was decreased in the striatum. The percentage of splenic T cells and B cells was not affected by 6-OHDA treatment. Although there is a marked increase in plasma corticosterone levels in 6-OHDA-treated mice, saline-injected control animals have equivalent increases in plasma corticosterone without concomitant impairment of the immune response. Thus, the decline in immune responsiveness following 6-OHDA treatment does not result from corticosterone-induced immunosuppression. Analysis of the kinetics of the primary IgG response following 6-OHDA treatment indicates that the magnitude, but not the kinetics, of the response decreases. Experiments to determine the effects of 6-OHDA on the afferent and efferent phrases of the response demonstrate that it is effective only when administered prior to immunization, and thus must inhibit early events involved in the initiation of the response. Additional experiments show that mice can be immunized 2 weeks following brain catecholamines depletion and still exhibit a decreased antibody response. However, the response returns to normal levels if immunization is delayed 4 weeks after injection. Further experiments demonstrated that 6-OHDA treatment has no effect on the secondary antibody response, but does inhibit the development of immunological memory. Collectively, these results indicate that 6-OHDA treatment has a profound inhibitory effect on the induction of the primary antibody response and immunological memory development, but is without effect on the secondary antibody response. The data further substantiate the existence of a link between the brain and the immune response.
We have examined the characteristics of a rat suppressor T cell (Ts) that inhibited the antigen-dependent proliferative response of antigen-primed T cells. The kinetics of in vitro induction of Ts from lymph node T cells obtained from antigen-primed rats indicated that Ts were induced in the presence of the priming antigen within 48 hr of culturing. The Ts produced during the first 48 hr of in vitro cultures were radiosensitive (2000 rad) but became partially radioresistant within the next 48 hr of culturing. In the presence but not the absence of priming antigen, Ts inhibited the antigen-dependent proliferative response to the priming antigen as well as to heterologous antigens. Suppression appeared to be mediated via a nondialyzable suppressor factor (TsF). The induction of Ts in cultures required the presence of OX-6-/OX-8- T cells, antigen-presenting cells, and the antigen. Although a majority of cells recovered from the induced cultures were OX-8+, there was no evidence that OX-8+ antigen expression per se was related to Ts activity. Addition of highly purified IL 2 augmented the Ts-mediated suppression. The immunoregulatory implications of these findings are discussed.
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The present study investigates the influence of pharmacological agents known to regulate biosynthesis of the neurotransmitter, serotonin (5-hydroxytryptamine, 5-HT) on the primary antibody response to sheep red blood cells (SRBC) in the CBA mouse. Systemic administration of 5-HT (4-100 mg/kg) or its precursor, 5-hydroxytryptophan (5-HTP, 50-400 mg/kg), 30-60 min before immunization resulted in dose-dependent suppression of both the IgM and IgG plaque-forming cell (PFC) response to SRBC. Conversely, para-chlorophenylalanine (PCPA, 250 mg/kg), which inhibits the rate-limiting enzyme (tryptophan hydroxylase) in 5-HT biosynthesis, markedly enhanced IgM and IgG antibody production when injected 48 hr prior to antigen. Effects of these drugs on immune processes appeared independent of observed changes in plasma corticosterone levels. Further, immune function was preserved following selective depletion of brain serotonin through intracisternal injection of the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) in mice pretreated with desmethylimipramine (DMI). Thus, immunomodulation by serotonin appears to be mediated via peripheral mechanism(s).