Compositional variation of the essential oils of Artemisia afra Jacq. from three provinces in South Africa--a case study of its safety.

Safety of Artemisia afar has been a controversial issue due to its high thujone content. Despite the declaration of the World Health Organization in the 1970s of the plant being unsafe for consumption, it is still commonly used in folklore medication in South Africa, especially in winter. Essential oils were isolated by hydrodistillation from the twigs of A. afra plants from different locations in the Eastern Cape, Free State and KwaZulu-Natal. Analyses of the oils by GC and GCMS revealed compositional variations in the levels of alpha-and beta-thujone, 1,8-cineole and camphor. alpha-Thujone was the major component of the essential oils ofA. afra from Philippolis (Free State) and Keiskammahoek (Eastern Cape) (62-74%), while the camphor content was very low (< or = 0.1-0.6%). The samples from Gqumahshe, Hogsback (Eastern Cape) and Empangeni (KwaZulu Natal) had low a-thujone contents (3.7-20.0%) while 1,8-cineole (13.0-49.5%) and camphor (13.9-21.2%) were the main components of the essential oils. It was further observed that the concentration of alpha-thujone increased significantly in the dry leaves when compared with the fresh leaves. This implies that fresh leaves are better used for infusion than dry leaves. This study reveals that not all A. afra contain high concentrations of alpha- and beta-thujone.

Sodium nitrite therapy attenuates the hypertensive effects of HBOC-201 via nitrite reduction.

Hypertension secondary to scavenging of NO remains a limitation in the use of HBOCs (haemoglobin-based oxygen carriers). Recent studies suggest that nitrite reduction to NO by deoxyhaemoglobin supports NO signalling. In the present study we tested whether nitrite would attenuate HBOC-mediated hypertension using HBOC-201 (Biopure), a bovine cross-linked, low-oxygen-affinity haemoglobin. In a similar way to unmodified haemoglobin, deoxygenated HBOC-201 reduced nitrite to NO with rates directly proportional to the extent of deoxygenation. The functional importance of HBOC-201-dependent nitrite reduction was demonstrated using isolated aortic rings and a murine model of trauma, haemorrhage and resuscitation. In the former, HBOC-201 inhibited NO-donor and nitrite-dependent vasodilation when oxygenated. However, deoxygenated HBOC-201 failed to affect nitrite-dependent vasodilation but still inhibited NO-donor dependent vasodilation, consistent with a model in which nitrite-reduction by deoxyHBOC-201 counters NO scavenging. Finally, resuscitation using HBOC-201, after trauma and haemorrhage, resulted in mild hypertension ( approximately 5-10 mmHg). Administration of a single bolus nitrite (30-100 nmol) at the onset of HBOC-201 resuscitation prevented hypertension. Nitrite had no effect on mean arterial pressure during resuscitation with LR (lactated Ringer's solution), suggesting a role for nitrite-HBOC reactions in attenuating HBOC-mediated hypertension. Taken together these data support the concept that nitrite can be used as an adjunct therapy to prevent HBOC-dependent hypertension.

Resuscitation from hemorrhagic shock with HBOC-201 in the setting of traumatic brain injury.

Outcomes after mild or moderate head trauma are worsened with associated hypotension, and secondary brain injury can be reduced with timely resuscitation. This study was performed to investigate HBOC-201 as a resuscitation therapy in a combined hemorrhagic shock and brain injury model. Anesthetized rats sustained moderate brain injury using a controlled cortical impact device, followed by rapid hemorrhage to a mean arterial pressure of 30 mmHg. After 30 min of hypotension, animals were resuscitated with HBOC-201, autologous shed blood (SB), or lactated Ringer solution (LR). Brain injury was assessed by measurements of cerebral blood flow (CBF) and cerebral vasoreactivity to hypercapnia (CVH) using a laser Doppler flowmeter. Contusion volume was evaluated histologically, and cerebral edema was determined by total water content. The HBOC rats required significantly less resuscitation volume versus LR and SB. The CBF was significantly diminished at 60 min after resuscitation with HBOC (70.1% +/- 3.8% baseline) compared with LR (105.8% +/- 10.1% baseline; P < 0.01) and SB (96.8% +/- 5% baseline; P < 0.05). The CVH was preserved in the HBOC and SB groups. The CVH was significantly diminished compared with baseline in the LR group at 30 min after resuscitation and showed a significant loss compared with HBOC at 60 min after resuscitation. The contusion volume for HBOC (45.1 mm3) and SB (35.1 mm3) was less than LR (63.5 mm3, P < 0.01). Although CBF was diminished after resuscitation in the HBOC group, HBOC-treated animals maintained CVH and experienced significantly smaller contusion volume than those treated with LR. These results suggest that resuscitation with HBOC-201 protects autoregulatory mechanisms and may reduce secondary brain injury in traumatic brain injury.

Phenotypic and functional characterization of long-term cultured rhesus macaque spleen-derived NKT cells.

Natural killer T cells are immunoregulatory cells, which have important roles in tolerance and autoimmunity, as demonstrated primarily in mice and humans. In this study, we define the phenotype and function of Valpha24(+) T cells derived from the spleens of rhesus macaques, a species increasingly used in models of immune tolerance. Valpha24(+) cells were isolated and expanded with monocyte-derived immature dendritic cells in the presence of alpha-galactosylceramide, IL-2, and IL-15. Rhesus NKT cells were stained with mAbs against both Valpha24 and the invariant complementarity-determining region 3 epitope of the human Valpha24/JalphaQ TCR. The cells were CD4, CD8 double negative and expressed CD56. Rhesus NKT cells also exhibited moderate to high expression of CD95, CD45RO, CD11a, and beta(7) integrin, but did not express CD45 RA, CD62L, CCR7, CD28, and other activation, costimulatory molecules (CD69 and CD40L). By intracellular staining, >90% of unstimulated rhesus NKT cells expressed IL-10, but not IFN-gamma. However, the latter was strongly expressed after stimulation. Rhesus NKT secreted large amounts of TGF-beta, IL-13, and IL-6, and modest levels of IFN-gamma, whereas IL-10 secretion was negligible and no detectable IL-4 was observed either intracellularly or in culture supernatants. Functionally, the NKT cells and their supernatants suppressed T cell proliferation in allogeneic MLR. We conclude that long-term cultured rhesus macaque spleen-derived Valpha24(+) T cells are semi-invariant double-negative cells with effector memory phenotype. These cells are semianergic, polarized to a uniquely Th3 > T regulatory-1 regulatory cell phenotype, and have regulatory/suppressive function in vitro.

The immune decision toward allograft tolerance in non-human primates requires early inhibition of innate immunity and induction of immune regulation.

Brief treatment of rhesus macaques with immunotoxin plus 15-deoxyspergualin has yielded exceptional numbers (54%) of stable tolerant kidney allograft recipients, surviving over 6 years without rejection or immunosuppression. An early increase in IL-10 and reduction in IFNgamma distinguished recipients that subsequently became tolerant. Furthermore, analysis suggested that this immune switch was programmed within hours of transplantation. Administering deoxyspergualin within 5 h of surgery gave a higher incidence of tolerance (76%) compared to administration >5 h before or after surgery (11%, P<0.01). Deoxyspergualin inhibits nuclear translocation of activated NF-kappaB through heat shock proteins. Lymph node biopsies from tolerant recipients showed significant reductions in cytoplasmic expression of Hsp70 and RelB and almost complete inhibition of nuclear translocation of both. The early timing effect of deoxyspergualin suggests a crucial limitation to induction of stable tolerance is activation of Hsp-dependent innate responses to damage by ischemia-reperfusion. This was supported by studies in murine kidney reperfusion injury, where deoxyspergualin given 5 h before reperfusion protected renal function and reduced levels of IL-6 and IL-12. The narrow timing window for initiating deoxyspergualin treatment suggests the innate immune system is poised to defeat allograft tolerance induction, so effective blockade of NF-kappaB-mediated innate immunity must be in place early, to enable development of a tolerogenic environment.

Transplantation: tolerance.

The immunosuppressive regimens currently in use have been responsible for major improvements in transplanted organ acceptance, but long-term survival can be compromised by drug toxicity and/or chronic immune deficiency. The ultimate goal for transplantation is tolerance, defined as durable, donor-specific allograft acceptance in the absence of long-term immunosuppression. This review describes current experimental strategies for tolerance induction in primate models that are poised for clinical application.

STEALTH in transplantation tolerance.

Although contemporary immunosuppressive regimens are responsible for major improvements in allograft acceptance, there are indications that long-term survival may be compromised through drug toxicity and/or chronic immune deficiency. The ultimate goal for transplantation is tolerance, defined as durable, donor-specific allograft acceptance in the absence of long-term immunosuppression. This article reviews the nonhuman primate STEALTH model of tolerance recently developed by the transplant immunobiology group at University of Alabama at Birmingham. The STEALTH model was designed for future application to human transplantation and comprises a concise peritransplant treatment strategy of only 2 wk. Tolerance is induced by depletion of T cells, with concomitant inhibition of nuclear factor-kappaB/RelB-dependent proinflammatory signaling. This treatment has resulted in an unprecedented frequency of kidney allograft survival (62.5% at 3 yr), with some primate recipients remaining in good health more than 6 yr posttransplant, in the complete absence of chronic pharmacologic immunosuppression.

Islet transplantation in the twenty-first century.

Isolated islet transplantation is poised for clinical application to treat insulin-dependent diabetes. Unlike exogenous insulin therapy, islet transplantation has promise for preventing and/or reversing the dismal secondary complications of diabetes. Islet transplants are arguably the most unique type of allografts, and we discuss their properties, limitations, and potential in this overview. The induction of immunologic tolerance to allow islet grafts to endure and prevail, without the hardship of chronic immunosuppressive therapy, is a major goal in this field. In this context, we discuss our successful results in preclinical models of primate allogeneic and xenogeneic islet graft tolerance.

Converting nonhuman primate dendritic cells into potent antigen-specific cellular immunosuppressants by genetic modification.

T cell depletion plus donor bone marrow cell (BMC) infusion induces long-term kidney allograft survival in a limited number of rhesus macaque recipients. Therefore, there is a need to enhance the tolerogenic activity of donor BMCs. The tolerogenic effect of donor BMCs is ascribed to a veto activity, mediated by a CD8+ subset that upregulates immunoregulatory effector molecules, transforming growth factor-beta1 (TGF-beta), and FasL, after interaction with donor-reactive cytotoxic T lymphocyte precursors (CTLp), leading to clonal inactivation/deletion of donor-reactive CTLp. Of note, the receptors for TGF-beta1- and FasL-induced signal transduction are upregulated in activated T cells. Since mature dendritic cells (DCs) are exceptionally efficient activators of T cells, we postulated that mature DCs modified to overexpress TGF-beta1 and FasL might exert potent veto (i.e., inactivating/deleting) activity independent of CD8 expression. A fusion protein comprising antihuman CD40 single-chain antibody and soluble coxsackie-adenovirus receptor enabled high-efficiency transduction of rhesus monocyte-derived DCs (Rh MDDCs) by recombinant adenovirus (Ad). Mature Rh MDDCs transduced with Ad encoding active TGF-beta1 retained a mature phenotype yet exhibited potent alloantigen-specific cellular immunosuppression. Such modified MDDCs have the potential to promote tolerance induction to allografts in vivo.

Pediatric hemofiltration: Normocarb dialysate solution with citrate anticoagulation.

Fourteen children, newborn to 17 years of age, underwent continuous veno-venous hemofiltration with dialysis (CVVHD), using a new FDA-approved bicarbonate-based calcium-free dialysis solution (Normocarb) in combination with citrate anticoagulation. Dialysis prescription included use of the PRISMA system (Gambro, Lakewood, Colo., USA), with ACD-A (Baxter, Deerfield, Ill., USA) for anticoagulation and Normocarb (Dialysis Solution, Richmond Hills, Ontario, Canada) for dialysate. Diagnosis included 11 children with sepsis and 3 children with tumor lysis syndrome. Mean weight was 31.6+/-4.7 kg (range 3.7-62 kg) and average length of therapy was 11.4+/-3.7 days (range 6 h to 67 days). Length of circuit patency was 71.3+/-7.2 h (range 6 h to 127 h), which was influenced in part by a decision to change circuits at 72 h as per manufacturer's recommendation. No bleeding occurred. This protocol utilizes industry-manufactured CVVHD machinery with both thermic and ultrafiltration control, with an effective anticoagulation protocol, and industry-produced bicarbonate dialysate. The use of industry machinery and solutions allows for consistent industrial quality assurance standards. This potentially may decrease the cost of therapy and minimize the risk of pharmacy errors that can occur with pharmacy-made dialysis solutions.

Absolute configurations of all four stereoisomers of cryptocaryalactone and deacetyl cryptocaryalactone.

Using information available in the literature and correlating this with physical data obtained for cryptocaryalactone stereoisomers isolated from C. myrtifolia and C. wyliei, it has been possible to assign the absolute configurations of the four stereoisomers of the title compound and its deacetyl analogue.