Establishment of HSV1 latency in immunodeficient mice facilitates efficient in vivo reactivation.

The establishment of latent infections in sensory neurons is a remarkably effective immune evasion strategy that accounts for the widespread dissemination of life long Herpes Simplex Virus type 1 (HSV1) infections in humans. Periodic reactivation of latent virus results in asymptomatic shedding and transmission of HSV1 or recurrent disease that is usually mild but can be severe. An in-depth understanding of the mechanisms regulating the maintenance of latency and reactivation are essential for developing new approaches to block reactivation. However, the lack of a reliable mouse model that supports efficient in vivo reactivation (IVR) resulting in production of infectious HSV1 and/or disease has hampered progress. Since HSV1 reactivation is enhanced in immunosuppressed hosts, we exploited the antiviral and immunomodulatory activities of IVIG (intravenous immunoglobulins) to promote survival of latently infected immunodeficient Rag mice. Latently infected Rag mice derived by high dose (HD), but not low dose (LD), HSV1 inoculation exhibited spontaneous reactivation. Following hyperthermia stress (HS), the majority of HD inoculated mice developed HSV1 encephalitis (HSE) rapidly and synchronously, whereas for LD inoculated mice reactivated HSV1 persisted only transiently in trigeminal ganglia (Tg). T cells, but not B cells, were required to suppress spontaneous reactivation in HD inoculated latently infected mice. Transfer of HSV1 memory but not OVA specific or naïve T cells prior to HS blocked IVR, revealing the utility of this powerful Rag latency model for studying immune mechanisms involved in control of reactivation. Crossing Rag mice to various knockout strains and infecting them with wild type or mutant HSV1 strains is expected to provide novel insights into the role of specific cellular and viral genes in reactivation, thereby facilitating identification of new targets with the potential to block reactivation.

Analysis of clinical and dermoscopic features for basal cell carcinoma neural network classification.

BACKGROUND: Basal cell carcinoma (BCC) is the most commonly diagnosed cancer in the USA. In this research, we examine four different feature categories used for diagnostic decisions, including patient personal profile (patient age, gender, etc.), general exam (lesion size and location), common dermoscopic (blue-gray ovoids, leaf-structure dirt trails, etc.), and specific dermoscopic lesion (white/pink areas, semitranslucency, etc.). Specific dermoscopic features are more restricted versions of the common dermoscopic features. METHODS: Combinations of the four feature categories are analyzed over a data set of 700 lesions, with 350 BCCs and 350 benign lesions, for lesion discrimination using neural network-based techniques, including evolving artificial neural networks (EANNs) and evolving artificial neural network ensembles. RESULTS: Experiment results based on 10-fold cross validation for training and testing the different neural network-based techniques yielded an area under the receiver operating characteristic curve as high as 0.981 when all features were combined. The common dermoscopic lesion features generally yielded higher discrimination results than other individual feature categories. CONCLUSIONS: Experimental results show that combining clinical and image information provides enhanced lesion discrimination capability over either information source separately. This research highlights the potential of data fusion as a model for the diagnostic process.

The endogenous cannabinoid anandamide shares discriminative stimulus effects with ∆(9)-tetrahydrocannabinol in fatty acid amide hydrolase knockout mice.

The endogenous cannabinoid system has been noted for its therapeutic potential, as well as the psychoactivity of cannabinoids such as Δ9-tetrahydrocannabinol (THC). However, less is known about the psychoactivity of anandamide (AEA), an endocannabinoid ligand. Thus, the goals of this study were to establish AEA as a discriminative stimulus in transgenic mice lacking fatty acid amide hydrolase (i.e., FAAH -/- mice unable to rapidly metabolize AEA), evaluate whether THC or oleamide, a fatty acid amide, produced AEA-like responding, and assess for CB(1) mediation of AEA's discriminative stimulus. Mice readily discriminated between 6mg/kg AEA and vehicle in a two-lever drug discrimination task. AEA dose-dependently generalized to itself. THC elicited full AEA-like responding, whereas oleamide failed to substitute. The CB(1) antagonist rimonabant attenuated AEA- and THC-induced AEA-appropriate responding, demonstrating CB(1) mediation of AEA's discriminative stimulus. These findings suggest that, in the absence of FAAH, AEA produces intoxication comparable to THC, and consequently to marijuana.

Reactive oxygen and reactive nitrogen as signaling molecules for caspase 3 activation in acute cardiac transplant rejection.

Apoptosis is a significant factor in cardiac dysfunction and graft failure in cardiac rejection. In this study, we examined potential signaling molecules responsible for caspase 3 activation in a model of acute cardiac allograft rejection. The roles of reactive oxygen species (ROS) and nitric oxide (NO) were determined in untreated allografts and allograft recipients treated with either cyclosporine (CsA), alpha-phenyl-t-butylnitrone (PBN, a spin-trapping agent), vitamin C (VitC), Mn(III)tetrakis (1-methyl-4-pyridyl)porphyrin); MnTmPyP, a superoxide dismutase (SOD) mimetic), or L-(1-iminoethyl)lysine) (L-NIL), an inhibitor of inducible NO synthase (iNOS) enzyme activity. Graft tissue was taken for measuring superoxide radical production, Western blotting, and direct measurement of caspase 3 activity. Activation of caspase 3 in untreated allografts was revealed by the appearance of cleaved caspase 3 from pro-caspase 3 by Western blotting and functional caspase 3 catalytic activity. CsA or PBN inhibited iNOS expression and caspase 3 activity. VitC and MnTmPyP did not alter iNOS expression or decrease NO levels but did inhibit caspase 3 activity. In contrast, L-NIL completely inhibited the increase in NO production without altering iNOS expression and inhibited caspase 3 activity. The prevention of TUNEL staining by MnTmPyP and L-NIL confirmed downstream effects of superoxide and NO on apoptosis. These studies indicate that both superoxide and NO (precursors of peroxynitrite formation) play a significant role in caspase 3 activation in cardiac allograft rejection.

Beneficial effect of alpha-tocopheryl succinate in rat cardiac transplants.

BACKGROUND: Anti-oxidant vitamins have increasingly been used to supplement traditional post-surgical treatment in cardiac transplant recipients. However, the mechanism(s) of action have not been determined. In this study we examined the effects of a novel vitamin E analog, alpha-tocopheryl polyethylene glycol-100 succinate (alpha-TPGS), and low-dose cyclosporine (CsA) in the treatment of acute and delayed cardiac rejection. METHODS: In situ sonomicrometry, histologic rejection and graft survival were determined in untreated rat cardiac allograft recipients and recipients receiving CsA, alpha-TPGS or CsA plus alpha-TPGS. DNA binding of nuclear factor (NF)-kappaB and AP-1, inducible nitric oxide synthase (iNOS) protein, caspase-3 activity and lymphocyte proliferation were determined. RESULTS: alpha-TPGS significantly (p < 0.05) prolonged graft survival equipotent to low-dose CsA. Treatment with CsA plus alpha-TPGS further enhanced graft survival (p < 0.001). CsA or alpha-TPGS alone decreased rejection, with the greatest decrease seen using combination therapy. Graft fractional shortening was improved by CsA or alpha-TPGS alone (p < 0.01), whereas distention in systolic and diastolic lengths in untreated allografts was prevented by CsA, alpha-TPGS and combination therapy. Nitrosylation of heme protein was inhibited by alpha-TPGS and abolished by CsA or CsA plus alpha-TPGS. Expression of iNOS was decreased 50% by alpha-TPGS equipotent to CsA, but apparently via an NF-kappaB- and AP-1-independent pathway. Caspase-3 activity, an index of apoptosis, was increased only in untreated allografts. In addition, alpha-TPGS markedly inhibited mitogen-stimulated proliferation by both rat and human lymphocytes. CONCLUSIONS: alpha-TPGS has a significant effect in limiting lymphocyte proliferation and activation. This might explain the equipotent action of alpha-TPGS vs low-dose CsA and its action to potentiate graft survival and limit graft rejection and dysfunction.

Post-translational modification of manganese superoxide dismutase in acutely rejecting cardiac transplants: role of inducible nitric oxide synthase.

BACKGROUND: Nitration of a critical tyrosine residue in the active site of manganese superoxide dismutase (MnSOD) can lead to enzyme inactivation. In this study, we examined the effect of inducible nitric oxide synthase (iNOS) on MnSOD expression, activity and nitration in acutely rejecting cardiac transplants. METHODS: Lewis (isograft) or Wistar-Furth (allograft) donor hearts were transplanted into Lewis recipient rats. Some rats received L-N6-(1-iminoethyl) lysine (l-NIL), a specific iNOS inhibitor. Protein nitration was determined by immunohistochemical, Western blot and slot-blot analyses. MnSOD enzyme activity and gene expression were determined using Western, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoprecipitation techniques. RESULTS: MnSOD protein levels were decreased 50% by post-operative day 6 (POD 6), which was prevented by L-NIL. RT-PCR analysis indicated that this decrease could not be explained by any changes in MnSOD mRNA. MnSOD enzyme activity but not protein was decreased at POD 5 in untreated allografts. The loss of MnSOD activity at POD 5 was also prevented by L-NIL. Immunoreactive nitrotyrosine was apparent in untreated allografts at POD 6. Slot-blot analysis indicated that nitrotyrosine formation in allografts could be blocked by L-NIL. Nitration of MnSOD was evident upon immunoprecipitation of MnSOD followed by Western blotting for nitrotyrosine. CONCLUSIONS: These results suggest that the decreased MnSOD enzyme activity in acutely rejecting cardiac allografts can be attributed to a post-translational modification related to nitration arising via an iNOS-dependent pathway. This could be a potential major source of amplified oxidative stress in acute graft rejection.