Fully automated 5-plex fluorescent immunohistochemistry with tyramide signal amplification and same species antibodies.

The ability to simultaneously visualize the presence, abundance, location and functional state of many targets in cells and tissues has been described as a true next-generation approach in immunohistochemistry (IHC). A typical requirement for multiplex IHC (mIHC) is the use of different animal species for each primary (1°Ab) and secondary (2°Ab) antibody pair. Although 1°Abs from different species have been used with differently labeled species-specific 2°Abs, quite often the appropriate combination of antibodies is not available. More recently, sequential detection of multiple antigens using 1°Abs from the same species used a microwaving treatment between successive antigen detection cycles to elute previously bound 1°Ab/2°Ab complex and therefore to prevent the cross-reactivity of anti-species 2°Abs used in subsequent detection cycles. We present here a fully automated 1°Ab/2°Ab complex heat deactivation (HD) method on Ventana's BenchMark ULTRA slide stainer. This method is applied to detection using fluorophore-conjugated tyramide deposited on the tissue and takes advantage of the strong covalent bonding of the detection substrate to the tissue, preventing its elution in the HD process. The HD process was characterized for (1) effectiveness in preventing Ab cross-reactivity, (2) impact on the epitopes and (3) impact on the fluorophores. An automated 5-plex fluorescent IHC assay was further developed using the HD method and rabbit 1°Abs for CD3, CD8, CD20, CD68 and FoxP3 immune biomarkers in human tissue specimens. The fluorophores were carefully chosen and the narrow-band filters were designed to allow visualization of the staining under fluorescent microscope with minimal bleed through. The automated 5-plex fluorescent IHC assay achieved staining results comparable to the respective single-plex chromogenic IHC assays. This technology enables automated mIHC using unmodified 1°Abs from same species and the corresponding anti-species 2°Ab on a clinically established automated platform to ensure staining quality, reliability and reproducibility.

Osteopontin-c is a selective marker of breast cancer.

While the acquisition of invasiveness is a critical step in early stage breast carcinomas (DCIS), no established molecular markers reliably identify tumor progression. The metastasis gene osteopontin is subject to alternative splicing, which yields 3 messages, osteopontin-a, osteopontin-b and osteopontin-c. Osteopontin-c is selectively expressed in invasive, but not in noninvasive, breast tumor cell lines, and it effectively supports anchorage independence. We evaluated osteopontin-c as a biomarker. The RNA message for osteopontin-c was present in 16 of 20 breast cancers (80%), but was undetectable in 22 normal specimens obtained from reduction mammoplasty. In contrast, osteopontin-a RNA was expressed at various levels in all 20 breast cancers, 11 tumor-surrounding tissues and 21 normal samples. The splice variant osteopontin-b was present at barely detectable levels in 18 of 20 cancers and in 6 of 22 normal breasts. By immunohistochemistry, 66 of 69 normal breasts were negative, while 3 showed low level staining. Among the breast cancers, 43 of 56 cores (77%) stained positive for osteopontin-c. When correlated with tumor grade, the staining for osteopontin-c increased from grade 1 to grade 3. In a total of 178 breast specimens analyzed, osteopontin-c was present in 78% of cancers, 36% of surrounding tissues and 0% of normal tissues. Furthermore, osteopontin-c detects a higher fraction of breast cancers than estrogen receptor (ER), progesterone receptor or HER2. In conjunction, osteopontin-c, ER and HER2 reliably predict grade 2-3 breast cancer. Hence, osteopontin-c is a diagnostic and prognostic marker that may have value in a diagnostic panel together with conventional breast cancer markers.

Sirolimus therapy of focal segmental glomerulosclerosis is associated with nephrotoxicity.

To evaluate the safety and efficacy of sirolimus in treating patients with focal segmental glomerulosclerosis (FSGS), we performed a phase 2, open-label clinical trial. Inclusion criteria were adults and children 13 years and older with biopsy-proven idiopathic FSGS, proteinuria with protein of 3.5 g/d or greater while on angiotensin antagonist therapy, glomerular filtration rate (GFR) of 30 mL/min/1.73 m(2) or greater (>or=0.50 mL/s), and failure to achieve sustained remission with at least 1 immunosuppressive agent. Eligible patients received sirolimus doses adjusted to achieve trough levels of 5 to 15 ng/mL during the first 4 months and 10 to 20 ng/mL for the subsequent 8 months. The primary outcome was decrease in proteinuria, expressed as complete remission (protein < 0.3 g/d) or partial remission (protein >or= 50% decrease and <3.5 g/d). Six adult patients with FSGS were enrolled in the study; they had median disease duration of 4.0 years, mean age of 39 +/- 11 years, mean baseline Modification of Diet in Renal Disease-estimated GFR of 52 +/- 15 mL/min/1.73 m(2) (0.87 +/- 0.25 mL/s), and median baseline proteinuria with protein of 6.6 g/d (interquartile range, 4.2 to 9.4). Five patients had received cyclosporine. No patient experienced a complete or partial remission. Sirolimus therapy was stopped prematurely in 5 patients for the following reasons: (1) precipitous decrease in GFR in 4 patients after 7 to 9 months of therapy with a greater than 2-fold increase in proteinuria in 3 patients and (2) hypertriglyceridemia with triglyceride levels greater than 1,600 mg/dL (>18 mmol/L) at 5 months in 1 patient. Because of a rapid decrease in GFR with worsening proteinuria, the protocol was closed to further recruitment. We conclude that sirolimus may be associated with nephrotoxicity in some patients with FSGS, particularly those with prolonged disease duration and prior cyclosporine therapy.

Use of mycophenolate mofetil for chronic, refractory immune cytopenias in children with autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of apoptosis associated most often with heritable FAS mutations leading to lymphadenopathy, hypersplenism and chronic refractory autoimmune cytopenias. Mycophenolate mofetil (MMF) was used to treat cytopenias in 13 ALPS patients aged 9 months to 17 years from a cohort of 118 children (aged < 18 years) and 82 adults. Twelve responded for a median follow-up of 49 weeks (range 38-240 weeks), defined by maintenance of adequate blood counts and reduction in dosage or cessation of other immunosuppressive agents. This preliminary experience suggests that MMF may spare steroid usage in patients with ALPS-associated cytopenias.

Effect of solution viscosity on intramolecular electron transfer in sulfite oxidase.

Our previous studies have shown that the rate constant for intramolecular electron transfer (IET) between the heme and molybdenum centers of chicken liver sulfite oxidase varies from approximately 20 to 1400 s(-1) depending upon reaction conditions [Pacheco, A., Hazzard, J. T., Tollin, G., and Enemark, J. H. (1999) J. Biol. Inorg. Chem. 4, 390-401]. These two centers are linked by a flexible polypeptide loop, suggesting that conformational changes, which alter the Mo-Fe distance, may play an important role in the observed IET rates. In this study, we have investigated IET in sulfite oxidase using laser flash photolysis as a function of solution viscosity. The solution viscosity was varied over the range of 1.0-2.0 cP by addition of either polyethylene glycol 400 or sucrose. In the presence of either viscosogen, an appreciable decrease in the IET rate constant value is observed with an increase in the solvent viscosity. The IET rate constant exhibits a linear dependence on the negative 0.7th power of the viscosity. Steady-state kinetics and EPR experiments are consistent with the interpretation that viscosity, and not other properties of the added viscosogens, is responsible for the dependence of IET rates on the solvent composition. The results are consistent with the role of conformational changes on IET in sulfite oxidase, which helps to clarify the inconsistency between the large rate constant for IET between the Mo and Fe centers and the long distance (approximately 32 A) between these two metal centers observed in the crystal structure [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garnett, R. M., Rajagopalan, K. V., Enemark, J. H., and Rees, D. C. (1997) Cell 91, 973-983].