Stephen A. Brown

Effect of smoking on human natural killer cell activity

Natural killer (NK) cells play a central role in immune surveillance against tumors and viral infections. NK activity is depressed in patients who have a wide range of carcinomas, including carcinomas of the lung. Peripheral blood NK activity was measured in 22 nonsmokers, 15 light/moderate smokers, 12 heavy smokers, and 19 patients with carcinoma of the lung. Patients with carcinoma of the lung had marked depression in NK activity compared with nonsmokers. Light/moderate smokers had NK activity comparable to that of nonsmokers, whereas heavy smokers had marked depression in NK activity that was comparable to that of patients with carcinoma of the lung. These results suggest that smoking‐induced alterations in NK activity may have a role in the pathogenesis of smoking‐associated carcinoma of the lung.

Effects of NF-κB1 (p50) targeted gene disruption on ionizing radiation-induced NF-κB activation and TNFα, IL-1α, IL-1β and IL-6 mRNA expression in vivo

Purpose : To investigate the role of the NF- κB1 (p50) gene in ionizing radiation (IR)-induced NF- κB activation and TNF α, IL-1 α, IL-1 β and IL-6 mRNA expression in vivo. Materials and methods : NF- κB activation was analysed by the gel shift/supershift assay and the levels of TNF α, IL-1 α, IL-1 β and IL-6 mRNA were measured using RNase protection assay (RPA). Various tissues from BALB/c, B6,129P-Nfkb1 (NF- κB1 or p50 gene knockout, p50 -/-) and B6,129PF2 (wild-type, p50 +/+) mice were analysed before or after exposure to a lethal dose (8.5 Gy) of total-body γ-irradiation. Results : Exposure of BALB/c mice to total-body IR selectively activated NF- κB in the spleen, mesenteric lymph nodes (LN) and bone marrow (BM). Gel supershift assay using polyclonal antibodies against NF- κB p50, p65 or c-Rel protein revealed that the NF- κB p50 subunit is a critical component of the NF- κB complexes activated by IR in vivo. Discretely augmented TNF α, IL-1 α, IL-1 β and IL-6 mRNA expression was found in the spleen, LN and BM after BALB/c mice received IR. However, mice lacking the p50 gene (p50 -/-) showed a significant reduction in IR-induced activation of NF- κB and increases in TNF α, IL-1 α, IL-1 β and IL-6 mRNA expression, as compared with that of wild-type mice (p50 +/+) . Conclusions : The NF- κB p50 subunit is a critical component of the NF- κB complexes activated by IR and it plays an important role in mediating IR-induced TNF α, IL-1 α, IL-1 β and IL-6 mRNA expression in vivo.

Activation of peripheral δ2 opioid receptors increases cardiac tolerance to ischemia/reperfusion injury Involvement of protein kinase C, NO-synthase, KATP channels and the autonomic nervous system

AIMS This study aims to investigate the role of peripheral delta(2) opioid receptors in cardiac tolerance to ischemia/reperfusion injury and to examine the contribution of PKC, TK, K(ATP) channels and the autonomic nervous system in delta(2) cardioprotection. MAIN METHODS Deltorphin II and various inhibitors were administered in vivo prior to coronary artery occlusion and reperfusion in a rat model. The animals were monitored for the development of arrhythmias, infarct development and the effects of selected inhibitors. KEY FINDINGS Pretreatment with peripheral and delta(2) specific opioid receptor (OR) antagonists completely abolished the cardioprotective effects of deltorphin II. In contrast, the selective delta(1) OR antagonist 7-benzylidenenaltrexone (BNTX) had no effect. The protein kinase C (PKC) inhibitor chelerythrine and the NO-synthase inhibitor L-NAME (N-nitro-L-arginine methyl ester) also reversed both deltorphin II effects. The nonselective ATP-sensitive K+ (K(ATP)) channel inhibitor glibenclamide and the selective mitochondrial K(ATP) channel inhibitor 5-hydroxydecanoic acid only abolished the infarct-sparing effect of deltorphin II. Inhibition of tyrosine kinase (TK) with genistein, the ganglion blocker hexamethonium and the depletion of endogenous catecholamine storage with guanethidine reversed the antiarrhythmic action of deltorphin II but did not change its infarct-sparing action. SIGNIFICANCE The cardioprotective mechanism of deltorphin II is mediated via stimulation of peripheral delta(2) opioid receptors. PKC and NOS are involved in both its infarct-sparing and antiarrhythmic effects. Infarct-sparing is dependent upon mitochondrial K(ATP) channel activation while the antiarrhythmic effect is dependent upon TK activation. Endogenous catecholamine depletion reduced antiarrhythmic effects but did not alter the infarct-sparing effect of deltorphin II.

The manganese superoxide dismutase mimetic, M40403, protects adult mice from lethal total body irradiation

Abstract Over-expression of manganese superoxide dismutase (MnSOD) protects tissues from radiation. M40403 is a stable non-peptidyl mimetic of MnSOD that crosses cell membranes and is effective in reducing experimental inflammation. Male BALB/c mice were injected intraperitoneally (i.p.) and subcutaneously (s.c.) with M40403, 30 min before 6.5, 7.5 and 8.5 Gy total body irradiation (TBI). Whereas all control injected mice died after receiving 8.5 Gy TBI by day 17, 30 day survival of mice pre-treated i.p. with 40, 30, 20 or 10 mg/kg was 100%, 90%, 81% and 25%, respectively. The Dose Reduction Factor 50/30 for animals treated with 30 mg M40403 s.c. 30 min prior to TBI was 1.41. Decreased apoptosis of the large and particularly the small bowel and marked recovery of both lymphoid and hematopoietic tissues occurred in the M40403 pre-treated animals. M40403 is effective in reducing TBI-induced tissue destruction and has potential as a new radioprotective agent.

Radiation mitigation effect of cultured mushroom fungus Hirsutella Sinensis (CorImmune) isolated from a Chinese/Tibetan herbal preparation –Cordyceps Sinensis

Purpose: This study was carried out to test the hypothesis that the anti-oxidant and growth promoting properties of the cultured mushroom fungus Hirsutella sinensis (CorImmune) of Cordyceps sinensis mitigate radiation injury in mice. Materials and methods: BALB/c mice received total body irradiation (TBI) followed by treatment with CorImmune. The effect of CorImmune on lymphoid tissue, spleen and blood cells as well as survival and hematopoietic recovery was compared to normal saline treated controls. Results: CorImmune administered beginning 2 hours after a lethal dose of TBI significantly improved survival: 55% in the CorImmune group vs. 0% in the saline control (p < 0.0001). It increased normal leukocyte levels in a dose-dependent fashion. Animals treated with sub-lethal TBI and monitored for blood leukocyte recovery exhibited a return to normal baseline 3 weeks after TBI injury. In contrast, only 50% returned to normal baseline in the saline control group (p < 0.01). CorImmune also stimulated immune lymphocyte proliferation by nearly two-fold in a 3H-thymidine incorporation assay compared to controls (p < 0.01). Conclusions: CorImmune significantly increased animal survival after a lethal dose of radiation, accelerated leukocyte recovery and stimulated immune lymphocyte proliferation. We conclude that CorImmune is effective as a radiation mitigator when administered after radiation injury.

Absence of IL-23p19 in donor allogeneic cells reduces mortality from acute GVHD

The p19 dimer of interleukin 23 (IL-23) has been reported to have a major role in the pathogenesis of many experimental and clinical autoimmune diseases and may also have a prominent role in transplantation. We reasoned that deficiency of p19 in the allogeneic donor transplant might reduce the inflammation caused by acute GVHD (aGVHD). The major histocompatibility complex-2 (H2d) BALB/c mice were subjected to 8.5 Gy TBI, followed by transplantation with 10 × 106 BM and 2.5 × 106 spleen cells from H2d BALB/c, H2b C57Bl/6 (B6) or H2b p19−/− donors. In all, 75% of the p19−/− transplanted mice survived, compared with only 12.5% of the B6 transplanted mice. This superior survival is correlated with significantly less severe aGVHD, absence of p19 after transplantation, less upregulation of mRNA and lower serum levels of IL-17 as compared with the B6 transplants. TBI alone significantly upregulated transforming growth factor-β (TGF-β), IL-6 and p19 mRNA levels in host BALB/c mice, possibly providing the milieu to induce IL-17 in p19−/− donor cells. IL-22, another cytokine, the induction of which in T-helper 17 (Th17) cells is supported by p19, was upregulated in BALB/c hosts but not in transplanted B6 or p19 donor cells, and may not have had a major role in modifying aGVHD.

T10B9 monoclonal antibody: A short-acting nonstimulating monoclonal antibody that spares γδ T-cells and treats and prevents cellular rejection

T10B9.1A-31/MEDI-500 is a nonmitogenic immunoglobulin M kappa murine monoclonal antibody (mAb) directed against the alpha-beta (αβ) heterodimer of the T-lymphocyte receptor complex. The hybridoma was first produced by fusing spleen cells from BALB/C mice immunized with human peripheral blood T-lymphocytes with SP2/O-Ag14 mutant myeloma cells. The mAb is produced and purified using multistep ion exchange and molecular sieve chromatography protocols. T10B9 has been used successfully to treat acute cellular rejection in renal transplantation and as an immunosuppression induction agent in heart and simultaneous kidney-pancreas transplantation. Because T10B9 is nonmitogenic and causes minimal cytokine release, both treatment of rejection and induction of immunosuppression were accomplished with significantly fewer and milder untoward effects (cytokine release syndrome) than its comparator OKT3. Since T10B9 is directed against the αβ heterodimer of the CD3 epitope, it spares the gamma delta (γδ) region. These gamma delta (γδ) T cells have a unique role in the immune response controlling many serious human diseases and perhaps facilitating the development of immunologic tolerance. T10B9 has a relatively short duration of action, depleting T cells for only 10 to 14 days, unlike the protracted depletion seen with thymoglobulin and Campath-1H. There is no B-lymphocyte depletion with T10B9 as there is with both of the aforementioned reagents. The lack of prolonged lymphocyte depletion may account for less infection observed with T10B9 treatment.

Activation of peripheral delta opioid receptors increases cardiac tolerance to arrhythmogenic effect of ischemia/reperfusion.

OBJECTIVES The objective of this study was to investigate the role of peripheral μ, δ1, δ2, and nociceptin opioid receptors agonists in the regulation of cardiac tolerance to the arrhythmogenic effect of ischemia/reperfusion in rats. METHODS Anesthetized open-chest male Wistar rats were subjected to either 45 minutes of left coronary artery occlusion (phase 1a 10 minutes and phase 2b 35 minutes) and 2 hours of reperfusion in Experiment 1 or 10 minutes of ischemia and 10 minutes of reperfusion in Experiment 2. In Experiment 1, saline or vehicle controls and the mu-specific opioids dermorphin-H (Derm-H) and ([d-Ala2, N-Me-Phe4, Gly-ol5] enkephalin (DAMAGO); the delta-1-specific opioid d-Pen2,5enkephalin (DPDPE); nociceptin; and the delta-2-specific opioids deltorphin-II (Delt-II), Delt-Dvariant (Delt-Dvar), and deltorphin-E (Delt-E) were infused 15 minutes prior to ischemia. In Experiment 2, DPDPE, Delt-D, Delt-Dvar, and Delt-E were infused at 15 minutes prior to ischemia. The universal opioid receptor antagonist naltrexone, the peripherally acting antagonist naloxone methiodide, the selective δ1 antagonist 7-benzylidene naltrexone maleate, and the specific δ2 antagonist naltriben mesylate were infused 25 minutes prior to ischemia. RESULTS In Experiment 1, pretreatment with the μ opioids Derm-H and DAMGO, DPDPE, and nociceptin at all doses tested did not reduce the incidence of ischemia-induced arrhythmias compared to controls during 45 minutes of ischemia. The δ2 opioids Delt-II (0.12 mg/kg), Delt-Dvar (0.3 mg/kg), and Delt-E (0.18 mg/kg) all demonstrated significant antiarrhythmic effects at the 150 nmol/kg dose compared to saline or vehicle controls. Nine of 19 animals treated with Delt-II were tolerant without ventricular arrhythmias to the arrhythmogenic effect of ischemia during the first 10 minutes of ischemia (phase 1a) and 11 of 19 were without ventricular arrhythmias during the following 35 minutes of ischemia (phase 1b). Delt-II also decreased the incidence of premature ventricular contractions and ventricular tachycardia by almost half during phase 1a. Delt-II did not affect the incidence of ventricular fibrillation (VF). Pretreatment with Delt-Dvar and Delt-E completely blocked the incidence of VF in phase 1b. Delt-E also decreased premature ventricular contractions by 50%, and the incidence of ventricular tachycardia decreased over twofold in phase 1b of ischemia. There was no enhanced tolerance by any of the delta-2 opioids to the arrhythmogenic effect of reperfusion after long-term ischemia. In Experiment 2, after 10 minutes of ischemia and 10 minutes of reperfusion, Delt-II (0.12 mg/kg) reduced the incidence of premature ventricular contractions and ventricular tachycardia compared to controls, and completely blocked the incidence of VF following 10 minutes of reperfusion. Delt-Dvar and Delt-E were without effect, as was DPDPE following 10 minutes of reperfusion. The antiarrhythmic effect of Delt-II during 10 minutes of ischemia and 10 minutes of reperfusion was completely blocked by the peripherally acting opioid receptor inhibitor naloxone methiodide and the selective delta-2 opioid receptor inhibitor naltriben mesylate, but not by the selective delta-1 inhibitor 7-benzylidene naltrexone maleate. The antagonists alone had no effect on arrhythmogenesis. CONCLUSIONS Peripheral delta-2 opioid receptor activation by Delt-II, Delt-Dvar, and Delt-E enhanced cardiac tolerance to the arrhythmogenic effects of ischemia.

Cellular origin of ionizing radiation‐induced NF‐κB activation in vivo and role of NF‐κB in ionizing radiation‐induced lymphocyte apoptosis

Purpose: To investigate the cellular origin of ionizing radiation (IR)‐induced NF‐κB activation in vivo and the role of NF‐κB in IR‐induced lymphocyte apoptosis. Materials and methods: NF‐κB activities were analysed by gel shift/supershift assay in isolated murine T‐ and B‐cells, macrophages (Mϕ) and tissues from normal and T‐ and B‐cell‐deficient Rag1 mice with or without exposure to IR. IR‐induced lymphocyte apoptosis was determined by analysis of 3,3′‐dihexyloxacarbocyanine iodide (DiOC6) uptake, annexin‐V staining and the sub‐G0/1 population, or by TUNEL assay. Results: The results showed that IR activated NF‐κB in lymphocytes, including both T‐ and B‐cells, but failed to do so in Mϕ. Furthermore, T‐ and B‐cell‐deficient Rag1 mice exposed to IR exhibited a significant reduction in NF‐κB activation as compared with normal mice. Although NF‐κB1 (p50) gene knockout or NF‐κB decoy oligonucleotide treatment specifically inhibited IR‐induced lymphocyte NF‐κB activation, they had no significant effect on IR‐induced lymphocyte apoptosis. Conclusions: This finding suggests that lymphocytes are the main cellular origin of IR‐induced NF‐κB activation in vivo. However, NF‐κB activation has no significant effect on IR‐induced lymphocyte apoptosis.

Expression of TNFα by CD3+ and F4/80+ cells following irradiation preconditioning and allogeneic spleen cell transplantation

Summary:The pathogenesis of acute graft-versus-host disease (aGVHD) includes tumor necrosis factor-alpha (TNFα) expression by macrophages and T cells. However, the temporal comparison of donor vs host cells to TNFα expression in response to irradiation conditioning and alloreactivity has not been reported. This study compared intracellular TNFα expression in donor vs host spleen T cells and macrophages using a murine model of aGVHD. Total body irradiation conditioning alone resulted in increased frequency of F4/80+/TNFα+ cells, but no increase in CD3+/TNFα+ cells. Syngeneic transplantation resulted in an increased frequency of F4/80+/TNFα+ cells, while CD3+/TNFα+ cells increased on days 1 and 3 but declined on day 5. Allogeneic transplantation resulted in an increased frequency of donor CD3+/TNFα+ cells, while the frequency of host CD3+/TNFα+ cells declined. Similarly, donor F4/80+/TNFα+ cells also increased in frequency after allotransplantation, while the frequency of host F4/80+/TNFα+ cells was increased on day 1 and declined through days 3 and 5. In absolute cell numbers, CD3+/TNFα+ cells were greater than F4/80+/TNFα+ cells post allotransplantation. We conclude that (1) both donor and host CD3+ and F4/80+cells are present in the post transplant period and contribute to TNFα production and (2) in terms of frequency, the majority of TNFα producing cells in the spleen after allogeneic BMT are CD3+.