Ultraviolet-A1 irradiation therapy for systemic lupus erythematosus.

Systemic lupus erythematosus (lupus, SLE) is a chronic autoimmune disease characterized by the production of autoantibodies, which bind to antigens and are deposited within tissues to fix complement, resulting in widespread systemic inflammation. The studies presented herein are consistent with hyperpolarized, adenosine triphosphate (ATP)-deficient mitochondria being central to the disease process. These hyperpolarized mitochondria resist the depolarization required for activation-induced apoptosis. The mitochondrial ATP deficits add to this resistance to apoptosis and also reduce the macrophage energy that is needed to clear apoptotic bodies. In both cases, necrosis, the alternative pathway of cell death, results. Intracellular constituents spill into the blood and tissues, eliciting inflammatory responses directed at their removal. What results is "autoimmunity." Ultraviolet (UV)-A1 photons have the capacity to remediate this aberrancy. Exogenous exposure to low-dose, full-body, UV-A1 radiation generates singlet oxygen. Singlet oxygen has two major palliative actions in patients with lupus and the UV-A1 photons themselves have several more. Singlet oxygen depolarizes the hyperpolarized mitochondrion, triggering non-ATP-dependent apoptosis that deters necrosis. Next, singlet oxygen activates the gene encoding heme oxygenase (HO-1), a major governor of systemic homeostasis. HO-1 catalyzes the degradation of the oxidant heme into biliverdin (converted to bilirubin), Fe, and carbon monoxide (CO), the first three of these exerting powerful antioxidant effects, and in conjunction with a fourth, CO, protecting against injury to the coronary arteries, the central nervous system, and the lungs. The UV-A1 photons themselves directly attenuate disease in lupus by reducing B cell activity, preventing the suppression of cell-mediated immunity, slowing an epigenetic progression toward SLE, and ameliorating discoid and subacute cutaneous lupus. Finally, a combination of these mechanisms reduces levels of anticardiolipin antibodies and protects during lupus pregnancy. Capping all of this is that UV-A1 irradiation is an essentially innocuous, highly manageable, and comfortable therapeutic agency.

Dopaminergic Immunofluorescence Studies in Kidney Tissue.

The kidney is a highly integrated system of specialized differentiated cells that are responsible for fluid and electrolyte balance in the body. While much of today's research focuses on isolated nephron segments or cells from nephron segments grown in tissue culture, an often overlooked technique that can provide a unique view of many cell types in the kidney is slice culture. Here, we describe techniques that use freshly excised kidney tissue from rats to perform a variety of experiments shortly after isolating the tissue. By slicing the rat kidney in a "bread loaf" format, multiple studies can be performed on slices from the same tissue in parallel. Cryosectioning and staining of the tissue allow for the evaluation of physiological or biochemical responses in a wide variety of specific nephron segments. The procedures described within this chapter can also be extended to human or mouse kidney tissue.

Persistence of restricted CD4 T cell expansions in SIV-infected macaques resistant to SHIV89.6P superinfection.

Attempts to evaluate the protective effect of live attenuated SIV vaccine strains have yielded variable results depending on the route of immunization, the level of attenuation, the level of divergence between the vaccine candidate and the challenge. The protective mechanisms induced by these vaccines are still not well understood. In an effort to address whether the diversity of the CD4+ T cell repertoire in cynomolgus macaques plays a role in the immunological protection following SIVmacC8 infection, we have performed a longitudinal follow-up of the CD4 repertoire by heteroduplex tracking assay in macaques mock-infected or infected with either the attenuated SIVmacC8 or its homologous SIVmacJ5 and challenged with simian-human immunodeficiency virus (SHIV89.6P). Viral load and CD4 absolute counts were determined in these animals and the presence of SHIV89.6P virus in challenged animals was evaluated by PCR and serology. In all macaques that were protected against the challenging virus, we demonstrated a reduced diversity in the CD4+ TRBV repertoire and a few dominant CD4+ T cell clones during early primary infection. In contrast, CD4 TRBV repertoire in unprotected macaques remained highly diverse. Moreover, some of the CD4 T cell clones that were expanded during primary SIV infection re-emerged after challenge suggesting their role in protection against the challenging virus. These results underline the importance of maintaining the CD4 T cell repertoire developed during acute infection and point to the restriction of the CD4 response to the vaccine as a correlate of protection.

A phase I trial of the selective oral cyclin-dependent kinase inhibitor seliciclib (CYC202; R-Roscovitine), administered twice daily for 7 days every 21 days.

Seliciclib (CYC202; R-roscovitine) is the first selective, orally bioavailable inhibitor of cyclin-dependent kinases 1, 2, 7 and 9 to enter clinical trial. Preclinical studies showed antitumour activity in a broad range of human tumour xenografts. A phase I trial was performed with a 7-day b.i.d. p.o. schedule. Twenty-one patients (median age 62 years, range: 39-73 years) were treated with doses of 100, 200 and 800 b.i.d. Dose-limiting toxicities were seen at 800 mg b.i.d.; grade 3 fatigue, grade 3 skin rash, grade 3 hyponatraemia and grade 4 hypokalaemia. Other toxicities included reversible raised creatinine (grade 2), reversible grade 3 abnormal liver function and grade 2 emesis. An 800 mg portion was investigated further in 12 patients, three of whom had MAG3 renograms. One patient with a rapid increase in creatinine on day 3 had a reversible fall in renal perfusion, with full recovery by day 14, and no changes suggestive of renal tubular damage. Further dose escalation was precluded by hypokalaemia. Seliciclib reached peak plasma concentrations between 1 and 4 h and elimination half-life was 2-5 h. Inhibition of retinoblastoma protein phosphorylation was not demonstrated in peripheral blood mononuclear cells. No objective tumour responses were noted, but disease stabilisation was recorded in eight patients; this lasted for a total of six courses (18 weeks) in a patient with ovarian cancer.

Elimination of anticardiolipin antibodies and cessation of cognitive decline in a UV-A1-irradiated systemic lupus erythematosus patient.

In the first patient with high levels of anticardiolipin antibodies (aCL) treated with ultraviolet-A1 (UV-A1; 320-400 nm) radiation, eight months of twice-weekly low-dose (10 J/cm(2)) irradiation was accompanied by the decrease of aCL levels to normal, cessation of clinical and positron emission tomographic (PET) scanning evidence of cognitive decline, and reversal of livedo reticularis. All occurred within the framework of an improving Revised Systemic Lupus Activity Measure (SLAM-R) score. Further studies in systemic lupus erythematosus (SLE) patients with elevated aCL are indicated.

Reversal of brain dysfunction with UV-A1 irradiation in a patient with systemic lupus.

Low-dose ultraviolet A-1 (UV-A1; 340-400 nm) bodily irradiation significantly reduces clinical manifestations of systemic lupus erythematosus (SLE). As neuropsychiatric-like symptoms respond prominently, a single patient was selected to undergo positron emission tomography (PET) before and after therapy to determine the effects of the therapy on the brain. The functional changes in 18F-deoxyglucose uptake as determined by PET imaging in this SLE patient indicated that improvement in brain function paralleled the reversal of cognitive deficits noted after the administration 160 kJ of bodily UV-A1 irradiation administered three times a week. Also of interest is that the UV-A1 irradiation, for the first time, ameliorated discoid lupus rashes, presumably due to a systemic action, as the lesions were for the first time covered during therapy.

Genetic and functional analyses of FH mutations in multiple cutaneous and uterine leiomyomatosis, hereditary leiomyomatosis and renal cancer, and fumarate hydratase deficiency.

Germline mutations of the fumarate hydratase (FH, fumarase) gene are found in the recessive FH deficiency syndrome and in dominantly inherited susceptibility to multiple cutaneous and uterine leiomyomatosis (MCUL). We have previously reported a number of germline FH mutations from MCUL patients. In this study, we report additional FH mutations in MCUL and FH deficiency patients. Mutations can readily be found in about 75% of MCUL cases and most cases of FH deficiency. Some of the more common FH mutations are probably derived from founding individuals. Protein-truncating FH mutations are functionally null alleles. Disease-associated missense FH changes map to highly conserved residues, mostly in or around the enzyme's active site or activation site; we predict that these mutations severely compromise enzyme function. The mutation spectra in FH deficiency and MCUL are similar, although in the latter mutations tend to occur earlier in the gene and, perhaps, are more likely to result in a truncated or absent protein. We have found that not all mutation-carrier parents of FH deficiency children have a strong predisposition to leiomyomata. We have confirmed that renal carcinoma is sometimes part of MCUL, as part of the variant hereditary leiomyomatosis and renal cancer (HLRCC) syndrome, and have shown that these cancers may have either type II papillary or collecting duct morphology. We have found no association between the type or site of FH mutation and any aspect of the MCUL phenotype. Biochemical assay for reduced FH functional activity in the germline of MCUL patients can indicate carriers of FH mutations with high sensitivity and specificity, and can detect reduced FH activity in some patients without detectable FH mutations. We conclude that MCUL is probably a genetically homogeneous tumour predisposition syndrome, primarily resulting from absent or severely reduced fumarase activity, with currently unknown functional consequences for the smooth muscle or kidney cell.

Total mercury and methylmercury levels in some New Zealand commercial marine fish species.

Two groups of samples spanning 16 years are reported for methylmercury and total mercury. All the samples had been taken from commercial catches and represent 33 different commercially important New Zealand marine fish species. Results show the New Zealand fish species sampled have mean contents of total mercury that range between 0.02 and 2.48 mg kg(-1) and mean contents of methylmercury that range from less than 0.04 to 1.97 mg kg(-1).

Assessment of cancer risk from ethylene oxide residues in spices imported into New Zealand.

Quantitative estimates of cancer risks from ethylene oxide (ETO) residues were constructed based on 200 retail samples of various spices in New Zealand. Two samples of cinnamon contained detectable ETO. The highest value encountered was 15 ppm. ETO was not detected in the remaining 198 samples. However, 31 samples had detectable levels of ethylene chlorohydrin (ECH) and/or ethylene bromohydrin (EBH). A conservative estimate of ETO intake, based on average spice consumption, was 3.4 x 10(-6) mg/kg/day. Cancer potency factors for ETO ranging from 0.29 to 0.55 (mg/kg/day)(-1) were used to form cancer risk estimates. The resulting estimates of average lifetime excess cancer risk was 0.8 x 10(-6) to 1.7 x 10(-6). The US 97.5 percentile value for spice consumption (2.8 kg spices per year), gave an extreme upper-end estimate of lifetime cancer risk of approximately 1.4 x 10(-5). These risks are practically negligible considering the conservative assumptions used in estimating exposure to ETO. The exposures to ECH and EBH are 200-300-fold higher than to ETO. These compounds are of lesser potency to ETO in terms of mutagenicity or carcinogenicity in studies to date. However, the precise contribution of these compounds to the cancer risk estimate is uncertain due to large toxicological data gaps, including the absence of a 2-year cancer bioassay by the oral route.

Quantitative relationship between MHC class II-superantigen complexes and the balance of T cell activation versus death.

The binding of bacterial superantigens (SAgs) is profoundly affected by the nature of the MHC class II-associated antigenic peptide. It was proposed that this limitation in the density of SAgs displayed at the surface of APCs is important for efficient TCR serial triggering as well as for preventing apoptosis of the responding T lymphocytes. Here, we have addressed quantitatively the size of this SAg-receptive pool of HLA-DR molecules that are available to bind and present staphylococcal enterotoxin A (SEA) at the surface of B lymphocytes. Our binding curves, depletion experiments, and quantitative immunoprecipitations show that about half the HLA-DR class II molecules on B cells are refractory to SEA binding. Yet, as compared with typical nominal Ags, an unusually high amount of class II-SAg complexes can be presented to T cells. This characteristic appears to be necessary for SAg-induced T cell apoptosis. When <0.3% of the total cell surface MHC class II molecules are occupied by SEA, T cells undergo a normal sequence of early activation events. However, presentation of a ligand density beyond this threshold results in T cell activation that is readily aborted by apoptosis but only after a few cell divisions. Thus, we confirm the existence of MHC class II subsets that are structurally unable to present SEA and provide a quantitative framework to account for the ability of bacterial SAgs to induce peripheral activation vs tolerance in the host.

Alternative proteolytic processing of mouse mammary tumor virus superantigens.

Mouse mammary tumor viruses express a superantigen essential for their life cycle. It has been proposed that viral superantigens (vSags) require processing by prohormone convertases (PCs) for activity. We now observe, using a panel of mutant forms of potential PC cleavage sites and in vitro cleavage assays, that only the CS1 (position 68 to 71) and CS2 (position 169 to 172) sites are utilized by furin and PC5. Other members of the convertase family that are expressed in lymphocytes are not endowed with this activity. Furthermore, mutant forms of two different viral superantigens, vSag7 and vSag9, which completely abrogated in vitro processing by convertases, were efficient in functional presentation to responsive T-cell hybridomas. This effect was observed in both endogenous presentation and paracrine transfer of the vSag. Processing by convertases thus appears not to be essential for vSag function. Finally, we have identified the purified endosomal protease cathepsin L as another protease that is able to cleave convertase mutant vSag in vitro, yielding fragments similar to those detected in vivo, thus suggesting that proteases other than convertases are involved in the activation of vSags.

Ultraviolet A1 (340-400 nm) irradiation and systemic lupus erythematosus.

Short wavelengths of ultraviolet (UV) light are clearly harmful in systemic lupus erythematosus (SLE), but the action of long UV wavelengths in SLE is more enigmatic. In a series of animal and human studies, long-wavelength UV radiation, i.e., radiation in the ultraviolet-A1 (UVA1) range (340-400 nm), has proven effective in the treatment of SLE. Disease amelioration and a marked decrease in mortality followed ultraviolet-A (UVA) radiation (320-400 nm) of the New Zealand White/New Zealand Black mouse model of lupus. A follow-up study in the same animal suggested that the longer wavelengths (UVA1, 340-400 nm) in the UVA wave band were primarily responsible. There followed four human studies. The first three of these provided data indicating that low-dose UVA1 radiation significantly reduced constitutional symptoms, joint pain, rashes, and the systemic lupus activity measures, a validated gauge of disease activity in SLE. The fourth human study showed that the therapeutic action of low-dose UVA1 action persisted or progressed long term, a period averaging 3.4 y. UVA1 effects on DNA repair, cell-mediated immunosuppression, tumor necrosis factor alpha release, and apoptosis contrast markedly with those of ultraviolet B (UVB, 280-320 nm) radiation and afford a possible basis for the salutary action of this modality of treatment. The unique features of UVA1 wavelengths may be suited to further therapeutic use, not only in SLE but also in other immunologic disorders.

Biomechanical coupling in renin-releasing cells.

The renin-angiotensin system is a major regulatory system controlling extracellular fluid volume and blood pressure. The rate-limiting enzyme in this hormonal cascade is renin, which is synthesized and secreted into the circulation by renal juxtaglomerular (JG) cells. The renal baroreceptor is a key physiologic regulator of renin secretion, whereby a change in renal perfusion pressure is sensed by these cells and results in a change in renin release. However, the mechanism, direct or indirect, underlying pressure transduction is unknown. We studied the direct application of mechanical stretch to rat JG cells and human renin-expressing (CaLu-6) cells on the release of renin. JG cells released a low level of baseline renin, comprising < 5% of their total renin content. By contrast, renin secretion from CaLu-6 cells comprised approximately 30% of cellular stores, yet was also stimulated twofold by 10 microM forskolin (P

Longterm ultraviolet-A1 irradiation therapy in systemic lupus erythematosus.

OBJECTIVE: In a recent series of short term studies ultraviolet-A1 (UV-A1; 340-400 nm) dermal irradiation proved effective in reducing signs and symptoms of disease activity in patients with systemic lupus erythematosus (SLE). To determine if the effectiveness persisted with longterm therapy, we followed the progress of 6 of these patients for an average of 3.4 (range 2.4-4.5) yrs. The 6 had had significant decreases in signs and symptoms of disease activity during the first 12 weeks of the earlier studies while receiving 3 to 5 low dose UV-A1 irradiations weekly and were asked to continue into longterm therapy. METHODS: Longterm therapy consisted of 1 or 2 irradiations of 6-15 J/m2 (15-30 min, or about 1/8-1/4 minimal erythema dose) per week. We assessed their progress every 3 mo with the systemic lupus activity measures. RESULTS: Despite the smaller number of weekly treatments, the gains achieved during the initial 12 weeks of the early studies not only persisted but increased slightly. Tanning was moderate to absent, the therapy was well tolerated, and there was no apparent toxicity. CONCLUSION: UV-A1 radiation induced remissions in SLE persist with longterm therapy; 1 or 2 weekly exposures suffice; there appears to be no significant toxicity.