Significant Increase In Staining Research Articles

Evaluating color stability and enamel surface roughness following resin infiltration treatment.

To assess the impact of resin infiltration treatment on enamel color stability and surface roughness after simulating daily exposure to coffee stains and regular toothbrushing with standard toothpaste. In this in vitro study, we utilized 47 caries-free human enamel specimens, which were divided into two distinct groups. The teeth in the study group underwent artificial demineralization, Icon resin infiltration, and polishing, while the control group received only polishing. Following this, all teeth from both groups were regularly immersed in freshly brewed coffee for 15 min, twice daily, over a span of 30 days. After each staining session, the teeth underwent 10 heavy circular strokes using a soft toothbrush and standard toothpaste. Color changes were measured on Days 1 and 30 using a spectrophotometer. The L*, a*, b* color system defined by the International Commission on Illumination was used to assess the changes. Surface roughness was evaluated at baseline and after 30 days using a 3D surface Metrology Microscope. After 30 days of immersion in coffee, both the study and control groups showed significant changes in color. However, differences were statistically significant between groups for "L" and "a" parameter but not for "b." The overall color change in the study group was higher than the control group. There were no significant differences in surface roughness within or between the groups before and after staining and brushing. Resin infiltration leads to a significant increase in staining compared to untreated enamel. These staining properties should be taken into account when making clinical decisions regarding the treatment of white spot lesions.

Primary blast injury causes cognitive impairments and hippocampal circuit alterations

Blast-induced traumatic brain injury (bTBI) and its long term consequences are a major health concern among veterans. Despite recent work enhancing our knowledge about bTBI, very little is known about the contribution of the blast wave alone to the observed sequelae. Herein, we isolated its contribution in a mouse model by constraining the animals' heads during exposure to a shockwave (primary blast). Our results show that exposure to primary blast alone results in changes in hippocampus-dependent behaviors that correspond with electrophysiological changes in area CA1 and are accompanied by reactive gliosis. Specifically, five days after exposure, behavior in an open field and performance in a spatial object recognition (SOR) task were significantly different from sham. Network electrophysiology, also performed five days after injury, demonstrated a significant decrease in excitability and increase in inhibitory tone. Immunohistochemistry for GFAP and Iba1 performed ten days after injury showed a significant increase in staining. Interestingly, a threefold increase in the impulse of the primary blast wave did not exacerbate these measures. However, we observed a significant reduction in the contribution of the NMDA receptors to the field EPSP at the highest blast exposure level. Our results emphasize the need to account for the effects of primary blast loading when studying the sequelae of bTBI.

A longitudinal study of CEACAM1 expression in melanoma disease progression.

The present study characterized the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) expression profile in a longitudinal study during melanoma progression, in lesions obtained from the same patients: a primary skin lesion, a lymph node and a distant metastasis. The present study is expected to increase our understanding of the expression patterns of CEACAM1 in melanoma development. We identified 20 patients who could be analyzed for CEACAM1 expression over the course of disease progression. The pathology blocks were cut, and two slides were generated for each specimen. One underwent standard hematoxylin and eosin (H&E) staining and a corresponding slide underwent immunohistochemical staining for the detection of CEACAM1. For 13 patients who were able to be followed up serially from primary lesion, lymph node and distant metastasis, a borderline significant increase in the staining of the membrane was noted (P=0.06). In contrast, there was no equivalent increase in cytoplasmic CEACAM1 in the same group of patients. For the cohort of 20 patients with primary and distant metastasis, a significant increase in the membrane staining was noted (P=0.026) and again, no equivalent significant increase in cytoplasmic staining was observed. We report that CEACAM1 expression increases along the course of disease development and progression of a patient. CEACAM1 represents a novel area of research which may have profound influence in future methods of harnessing cellular immunity to combat this disease. The results of the present study confirm that CEACAM1 is potentially an extremely useful target in arresting melanoma progression.

Effect of excess iodide on the structure of the thyroid gland of prepubertal male albino rats

Introduction Excessive iodine intake is emerging as a new healthcare-related concern. Excess iodine is associated with a spectrum of effects on the thyroid. Aim of work The aim of this study was to assess the effects of excess potassium iodide (KI) on the histological and immunohistochemical structure of the thyroid gland and evaluate the biochemical changes in thyroid hormones. Materials and methods Forty prepubertal male albino rats aged between 4 and 6 weeks were classified into two equal groups: group 1 (the control group), which was given distilled water, and group 2 (the KI-treated group), which was further subdivided into two equal subgroups. Subgroup 1a was given 0.1 mg KI/kg/day for 4 successive weeks and subgroup 1b was given 0.1 mg KI/kg/day for 8 successive weeks. At the end of the period, the animals were anesthetized and blood samples were collected for measurement of serum T3, T4, and thyroid-stimulating hormone. Thyroid glands were dissected out and processed for examination using light and electron microscopes. For light microscopic examination, the sections were stained with H&E and immunohistochemical analysis was carried out for localization of Fas. Results H&E-stained sections of the KI-treated subgroups showed highly significantly enlarged follicular cavities that were distended with vacuolated colloid. Most of the follicles were lined with flattened follicular cells with flattened nuclei. Follicular epithelial height was highly significantly decreased. Ultrastructurally, follicular cells showed dilatation of the cisternae of the endoplasmic reticulum, mitochondrial swelling, many electron-lucent zones, and collagen fibers in the interstitial spaces. Also, most of the nuclei were heterochromatic. Immunohistochemically, there was a highly significant increase in staining for Fas in the follicular cells of the treated subgroups. Biochemical assessment detected a nonsignificant decrease in T3 and T4 and a highly significant increase in the thyroid-stimulating hormone levels of the treated group. Conclusion Excessive iodine intake affects thyroid structure and function. Hence, public iodine intake should be regulated to make sure that it is within optimal levels. Proper monitoring of the salt iodization program is essential. Also, drugs with high iodine content should be administered with caution.

Is chlorhexidine mouth rinse, used as a mono-therapy or adjunct with oral hygiene, effective at reducing plaque growth and gingival inflammation?

This study describes a systematic review of the best available evidence on the effectiveness of a chlorhexidine mouth rinse as a mono-therapy or as an adjunct to mechanical oral hygiene against plaque growth and gingival inflammation. Medline, EMBASE and the Cochrane Central register of Controlled Trials were searched up to April 2011. Randomised controlled clinical trials that compared chlorhexidine to placebo or controlled mouth rinses or regular oral hygiene for a minimum duration of at least four weeks amongst gingivitis patients (≥ 18 years of age) were included. A total of 30 publications fulfilled the selection criteria. Clinical parameters measured at baseline and end of trial were plaque, gingival inflammation, bleeding and staining. Chlorhexidine reduced plaque by 33% and gingivitis by 26% compared to a placebo or a control mouth rinse. The investigators concluded that when used together with oral hygiene, chlorhexidine mouth rinses provide significant reductions in plaque and gingivitis scores in gingivitis patients, but a significant increase in staining compared to placebo or control mouth rinses.

Color stability of five orthodontic clear elastic ligatures

In this study the color stability of five commercially available orthodontic clear elastic ligatures daily exposed to a staining agent (red wine) was investigated. The commercial brands tested were 3M Unitek, Abzil, American Orthodontics, Dentsply GAC, and Morelli. Baseline color readings (Commission Internationale de l'Eclairage [CIE] L*a*b* parameters) of 20 ligatures for each brand were carried out using a spectrophotometer. The ligatures were divided in two groups (n = 10): one group remained immersed in distilled water (control) throughout the period, while the other group was daily exposed to red wine (1 hour of immersion). Immersion media were daily renewed. Successive color readings were carried out after 7, 14, 21, and 28 days, and the chromatic variations calculated. Color variation data were analyzed by two-way repeated measures analysis of variance and Student-Newman-Keuls test (P < .05). Storage in water increased staining until 14 days, and color receded after this period, irrespective of the brand. After 28 days of immersion in water, no significant difference among the commercial brands was observed. For the groups exposed to red wine, a significant increase in staining over the course of time was detected, irrespective of the brand. Ligatures from GAC and Morelli showed higher staining than the other materials, irrespective of the evaluation time. Ligatures from 3M Unitek and American generally showed lower pigmentation than the other brands. Exposure to a staining agent, cumulative exposure time, and commercial brand are factors that influence the color stability of clear elastic ligatures.

Surface discoloration of composite resins: Effects of staining and bleaching

The purpose of this in vitro study was to evaluate surface discoloration of three microhybrid composite resins (Esthet•X HD, Clearfil AP-X, Gradia Direct) and five nanohybrid composite resins (Ceram•X, GC Kalore, G-aenial, Grandio, GrandioSO), after staining and bleaching procedures. The composite resins were polymerized with a curing light (Celalux II, Voco, Cuxhaven, Germany) into 160 silicon molds (6,4 mm in diameter and 2 mm in thickness) to obtain identical specimens. Twenty samples for each composite resin were prepared. The specimens were polished using an automated polishing machine with the sequence of 600-, 800-, 1000-grit abrasive paper under water irrigation. The specimens were immersed in tea and distilled water: the specimens were dipped for 20 min, once a day (every 24 h), for 14 days into the drinks. The specimens were then bleached with carbamide peroxide at 17% (Perfect Bleach-Voco). The color of specimens was measured with a spectrophotometer according to the CIE L(*)a(*)b(*) system after light-polymerization of composite resin specimens, after 7 days, after 14 days, and after bleaching. The color difference h index (DEab(*)) between each measurement was calculated. Statistical analysis was made using analysis of variance (ANOVA). All specimens showed a significant increase in staining with a similar trend and no significant differences between microhybrid and nanohybrid composite resins. After whitening procedures, materials tested showed both significant and unsignificant differences of the h index. Microhybrid and nanohybrid composite resins had similar in vitro surface discoloration in tea. After bleaching, discoloration was removed from some composite resins tested.

Regulation of tight junction proteins occludin and claudin 5 in the primate ovary during the ovulatory cycle and after inhibition of vascular endothelial growth factor

Ovarian follicular and corpus luteum development, including angiogenesis, are characterized by cell-cell rearrangements that may require dynamic changes in cell-cell adhesion. The present study investigates the expression of tight junction proteins occludin and claudin 5 during follicular and luteal development in the primate ovary and after inhibition of vascular endothelial growth factor (VEGF) by VEGF trap treatment. Occludin was localized to the plasma membrane of granulosa cells. During follicular development occludin staining decreased significantly (P < 0.05) and disappeared completely by the ovulatory stage. After inhibition of VEGF, occludin staining was significantly (P < 0.05) higher in the granulosa of secondary and tertiary follicles compared with controls. Claudin 5 was exclusively localized to the theca vasculature. A significant (P < 0.05) increase in staining was detected from the pre-antral to the antral and ovulatory stage. However, dual staining with CD31 revealed that within the theca endothelium the amount of claudin 5 remained constant during follicular development. Treatment with VEGF trap throughout the follicular phase revealed a lack of claudin 5 staining in the theca interna but no difference was observed in the remaining theca externa vasculature. In the corpus luteum, claudin 5 was also localized in the vasculature. Treatment with VEGF trap in the mid-luteal phase resulted in a significant increase in staining (P < 0.05). These results led us to hypothesize that tight junctions are involved in regulation of follicular growth, antrum transition and follicular angiogenesis which is compromised by VEGF inhibition. VEGF may influence luteal vascular permeability by regulation of the endothelial specific tight junction protein claudin 5.

Antibodies neutralizing Nogo-A increase pan-cadherin expression and motor recovery following spinal cord injury in rats

A rat model of spinal cord injury was used to test the hypothesis that Nogo-A monoclonal antibody (NEP1-40) promotes morphologic and functional recoveries of injured spinal cord. Nogo-A is a myelin-associated neurite outgrowth inhibitory protein, which blocks elongation nerve fibers and limits neuronal regeneration after central nervous system injury. Forty-four rats were utilized and allocated into control (vehicle) and NEP1-40-treated groups. In all animals, the spinal cord was hemi-transected at Th-10 and phosphate-buffered saline solution was immediately applied on the injured area in the control group. NEP1-40 solution was immediately applied on the hemi-transected area in the treatment group. Each group was subdivided into three subgroups according to the postsurgical day of killing (3, 8 and 21 days). The spinal cords were removed for analysis. Motor scores in the NEP1-40-treated groups were significantly higher than those in the vehicle groups both at 8 and 21 days post injury. Immunohistochemical staining for pan-cadherin, a marker of neuronal cell adhesion and axonal sprouting, revealed a significant increase in staining in the NEP1-40 treatment group at 8 and 21 days post injury. Transmission electron microscopical evaluation revealed degeneration of the myelin and loss of cytoarchitectural organization in the axons of controls. Better preservation and normal histologic features were observed in the NEP1-40-treated groups. We have demonstrated improved preservation of injured axons and significant pan-cadherin expression after NEP1-40 treatment after the spinal cord injury. Inhibition of Nogo-A may improve the capacity for neuronal regeneration after spinal cord injury.

Analyzing Patterns of Staining in Immunohistochemical Studies: Application to a Study of Prostate Cancer Recurrence

Immunohistochemical studies use antibodies to stain tissues with the goal of quantifying protein expression. However, protein expression is often heterogeneous resulting in variable degrees and patterns of staining. This problem is particularly acute in prostate cancer, where tumors are infiltrative and heterogeneous in nature. In this article, we introduce analytic approaches that explicitly consider both the frequency and intensity of tissue staining. Compositional data analysis is a technique used to analyze vectors of unit-sum proportions, such as those obtained from soil sample studies or species abundance surveys. We summarized specimen staining patterns by the proportion of cells staining at mild, moderate, and intense levels and used compositional data analysis to summarize and compare the resulting staining profiles. In a study of Syndecan-1 staining patterns among 44 localized prostate cancer cases with Gleason score 7 disease, compositional data analysis did not detect a statistically significant difference between the staining patterns in recurrent (n = 22) versus nonrecurrent (n = 22) patients. Results indicated only modest increases in the proportion of cells staining at a moderate intensity in the recurrent group. In contrast, an analysis that compared quantitative scores across groups indicated a (borderline) significant increase in staining in the recurrent group (P = 0.05, t test). Compositional data analysis offers a novel analytic approach for immunohistochemical studies, providing greater insight into differences in staining patterns between groups, but possibly lower statistical power than existing, score-based methods. When appropriate, we recommend conducting a compositional data analysis in addition to a standard score-based analysis.

Effect of retinoic acid in experimental diabetic nephropathy

Although the pathogenetic mechanism of diabetic nephropathy has not been elucidated, an inflammatory mechanism has been suggested to contribute to its progression. Monocyte chemoattractant peptide (MCP)-1 attracts macrophages and T cells, and ultimately injures renal tissue. In early diabetic nephropathy, urinary excretion of MCP-1 was elevated, and increased as renal damage became more severe. Podocytes are expected to have an inflammatory role in diabetic nephropathy, as the surface expression of chemokine receptors such as CCR and CXCR on these cells has been recently reported. Although retinoid (retinal), a known anti-inflammatory agent, has been reported to be beneficial in some experimental models of renal disease, it has not been determined to prevent disease progression in diabetic nephropathy. We investigated the effects of all-trans retinoic acid on the production of MCP-1 under high glucose conditions in cultured mouse podocytes. We also evaluated whether all-trans retinoic acid inhibits inflammatory changes and improves renal function during the early stages of diabetic nephropathy in streptozotocin-induced diabetic rats. In cultured podocytes, high glucose stimuli rapidly upregulated the MCP-1 mRNA transcript and protein release. Treatment with retinoic acid tended to suppress the MCP-1 gene transcript, and significantly inhibited MCP-1 protein synthesis induced by high glucose stimulation. Urinary protein excretion and the urinary albumin : creatinine ratio (ACR) were significantly higher in diabetic rats 4 weeks after the induction of diabetes mellitus compared with control rats, and retinoic acid treatment markedly decreased both proteinuria and urinary ACR (proteinuria: 1.25+/-0.69 vs 0.78+/-0.72 mg/mgCr, P=0.056; urinary ACR: 0.47+/-0.25 vs 0.21+/-0.06 mg/mgCr, P=0.088). Urinary excretion of MCP-1 was rapidly increased 2 days after induction of diabetes mellitus in diabetic rats, and further increased until rats were 4 weeks of age, compared with control rats. Retinoic acid treatment resulted in 30% reduction of the urinary level of MCP-1 compared with vehicle-treated diabetic rats (119.3+/-74.2 vs 78.1+/-62.7 pg/mgCr, P=0.078). Immunohistochemistry revealed a significant increase in staining for MCP-1 and anti-monocyte/macrophage (ED-1) protein in the diabetic kidney, and retinoic acid treatment significantly suppressed intrarenal MCP-1 and ED-1 protein synthesis. In conclusion, podocytes are involved in the inflammatory reaction under diabetic circumstances, and these reactions were suppressed by retinoic acid. Retinoic acid also suppressed inflammatory changes in the diabetic rat kidney, and decreased proteinuria in diabetic rats. These results suggest that retinoic acid may have renoprotective effects in the early stages of diabetic nephropathy through an anti-inflammatory activity.

Increases in amino-cupric-silver staining of the supraoptic nucleus after sleep deprivation

Sleep deprived rats undergo a predictable sequence of physiological changes, including changes in skin condition, increased energy expenditure, and altered thermoregulation. Amino-cupric-silver staining was used to identify sleep deprivation related changes in the brain. A significant increase in staining was observed in the supraoptic nucleus (SON) of the hypothalamus of rats with high sleep loss (>45 h) vs. their yoked controls. Follow-up experiments showed that staining was not significantly different in rats sleep deprived for less than 45 h, suggesting that injurious sleep deprivation-related processes occur above a threshold quantity of sleep loss. These anatomical changes suggest that the effects of sleep deprivation may be related to protein metabolism in certain brain regions.

Accessory cell populations in draining lymph nodes of lambs in the elicitation phase of DNCB-induced contact hypersensitivity

The effect of experimentally induced contact hypersensitivity on accessory cell populations in draining lymph nodes of lambs was studied. Previous studies of draining lymph nodes of lambs during the elicitation phase of CHS have shown that there are significant changes in T-cell subpopulations, particularly CD4 + cells and γδ T-cells, but the behaviour of accessory (antigen presenting) cell populations was not investigated. The immunohistochemical presence of accessory cell populations was determined using markers for CD68, Pan MHCII, MHCII DQ, MHCII DR, OvCD1w1 (putative human CD1a/c-like) and OvCD1w2 (human CD1b-like). Ten lambs were sensitised, and 14 days later re-challenged, by applying the hapten di-nitro-chloro-benzene (DNCB) together with an acetone and olive oil (AOO) vehicle, onto the skin. Cryosections of the draining lymph nodes were stained immunohistochemically for the accessory cell markers. Using an image analysis system, the areas of staining in the lymph nodes from the challenged animals were compared with measurements in control animals. A significant increase in staining for CD68 + cells was detected in the cortex of the DNCB-treated group ( p=0.003). A significant increase in staining for the Pan MHCII marker was also observed in the DNCB group ( p=0.013). These results show that MHCII + cells and CD68 + cells constitute a prominent cell population in the cortex of the regional lymph nodes of lambs in the late elicitation phase of DNCB-induced contact hypersensitivity.

Ancillary methods for the detection of recurrent urothelial neoplasia

Ancillary methods for the detection of recurrent urothelial neoplasia

Myofibroblast induction with transforming growth factor-β1 and −β3 in cutaneous fetal excisional wounds

Background/Purpose: In a noncontractile fetal rabbit model, the authors recently have shown the induction of excisional wound contraction with sustained-release cellulose implants formulated with transforming growth factor (TGF)-β. The purpose of this study was to test the hypothesis that the excisional wound contraction in this model is associated with the induction of myofibroblasts in the surrounding dermis, demonstrated by the presence of α-smooth muscle actin.Methods:Cellulose discs were formulated with either 1.0 μg of TGF-β1 (n = 6); 1.0 μg of TGF-β3 (n = 9); 10 μg of TGF-β3 (n = 6); or their carrier protein, bovine serum albumin (BSA; n = 9), for sustained-release over 5 days. Each disc was implanted into a subcutaneous pocket on the back of a fetal New Zealand White rabbit in utero on day 24 of gestation (term, 31 days). A full-thickness, 3-mm excisional wound (7.4 mm2) was then made next to the implanted cellulose disc. All fetuses were harvested at 3 days. The amount of α-smooth muscle (SM) actin in the dermis around the implants and wounds was determined using immunohistochemical techniques.Results: Excisional wounds exposed to 1.0 jig of TGF-β1 (5.6 ± 2.0 mm2), 1.0 μg of TGF-β3 (6.9 ± 1.0 mm2), and 10 μg of TGF-β3 (2.7 ± 1.0 mm2) were significantly smaller when compared with the BSA control group (12.8 ± 1.1 mm2; P < .05). Furthermore, there was a significant increase in staining for α-SM actin in the TGF-β1, (1.8 ± 0.5) and 10 μg TGF-β3 (2.8 ± 0.2) groups in comparison with the scant staining in the BSA control group (0.5 ± 0.2; P < .05).Conclusions:, TGF-β2 and −β3 induce α-SM actin and contraction of cutaneous excisional wounds in a fetal noncontractile model. This model of inducible cutaneous excisional wound contraction may be useful in further determining the role of the myofibroblast in wound contraction and the physiology underlying this poorly understood aspect of wound healing.

Expression of the adenocarcinoma-related antigen recognized by monoclonal antibody 44-3A6 in salivary gland neoplasias

The monoclonal antibody 44-3A6 detects a cell-surface protein that has been shown to be a useful marker in distinguishing adenocarcinomas from other histologic tumor types in a variety of tissues. The objective of this study was to determine whether 44-3A6 could be used as a tool in the classification of salivary gland neoplasms. These complex tumors share overlapping pathologic features but distinct clinical outcomes. This study used 44-3A6 to immunohistochemically describe the pattern and frequency of this antigen in salivary gland neoplasms. Formalin-fixed, paraffin-embedded tissue sections of 22 benign and 26 malignant salivary tumors were evaluated. The patient population consisted of 25 (52.1%) women and 23 (47.9%) men selected from archival pathology files to reflect a range of salivary gland diseases. Normal surrounding salivary glands were found to have intense focal staining almost exclusively localized to ductal luminal cells. There was little staining of either myoepithelial or acinar cells. A wide spectrum of expression was found between and within tumor types, but a trend toward more expression of this antigen with decreasing differentiation was seen. A significant increase in staining was also seen in those tumors with ductal differentiation ( n = 41) as opposed to those with predominantly acinar (i.e., acinic cell carcinoma) or myoepithelial (i.e., myoepithelioma; n = 8) differentiation (2.6 vs. 1.3, p < 0.05). No correlation was found between staining intensity and facial paralysis, pain, skin involvement, TNM stage, residual disease, or disease-free or total survival. Therefore this antigen appears to designate a duct luminal phenotype in normal and neoplastic salivary tissues. (Otolaryngol Head Neck Surg 1998;118:603-9.)

Expression of the adenocarcinoma-related antigen recognized by monoclonal antibody 44-3A6 in salivary gland neoplasias.

The monoclonal antibody 44-3A6 detects a cell-surface protein that has been shown to be a useful marker in distinguishing adenocarcinomas from other histologic tumor types in a variety of tissues. The objective of this study was to determine whether 44-3A6 could be used as a tool in the classification of salivary gland neoplasms. These complex tumors share overlapping pathologic features but distinct clinical outcomes. This study used 44-3A6 to immunohistochemically describe the pattern and frequency of this antigen in salivary gland neoplasms. Formalin-fixed, paraffin-embedded tissue sections of 22 benign and 26 malignant salivary tumors were evaluated. The patient population consisted of 25 (52.1%) women and 23 (47.9%) men selected from archival pathology files to reflect a range of salivary gland diseases. Normal surrounding salivary glands were found to have intense focal staining almost exclusively localized to ductal luminal cells. There was little staining of either myoepithelial or acinar cells. A wide spectrum of expression was found between and within tumor types, but a trend toward more expression of this antigen with decreasing differentiation was seen. A significant increase in staining was also seen in those tumors with ductal differentiation (n = 41) as opposed to those with predominantly acinar (i.e., acinic cell carcinoma) or myoepithelial (i.e., myoepithelioma; n = 8) differentiation (2.6 vs. 1.3, p < 0.05). No correlation was found between staining intensity and facial paralysis, pain, skin involvement, TNM stage, residual disease, or disease-free or total survival. Therefore this antigen appears to designate a duct luminal phenotype in normal and neoplastic salivary tissues.

CYCLOSPORINE RESISTANT CD28 CO-STIMULATORY PATHWAY INHIBITION WITH CURCUMIN MAY BE DUE TO APOPTOSIS

106 We have reported earlier that Curcumin (cur) possesses immunosuppressive properties in-vitro. It is also known to be a tyrosine kinase and protein kinase C inhibitor. It has been reported that cyclosporin A (CyA) does not inhibit CD28 co-stimlatory pathway of T cell activation. We hypothesized that Cur, a tyrosine kinase inhibitor, would block CyA resistant CD28 costimulatory pathway of human T cell proliferation, and that this effect may partially be due to increased apoptosis. Materials and Methods: Human T-lymphocytes were isolated from healthy donors using gradient centrifugation and rosetting techniques. In four separate experiments T cells were plated in triplicates in 96-well plates at a density of 2×105 cells/well. These cells were stimulated with 0.5 ng/ml phorbol myristate acetate (PMA) + 0.5 μg/ml anti-CD28 antibody (PMA-CD28 group) or 2.5 μg / ml PHA (PHA group). Cur or CyA at varying concentrations(0.31, 0.625, 1.25, 2.5, 5 or 10 μg/ ml and 1.25, 2.5, 5, 10, 20 or 250 ng/ ml respectively) was added and cellular proliferation was measured by the uptake of 3H thymidine and is reported (mean cpm/well±SD). Cells from PMA-CD28 group that were treated either with curcumin or 0.4% DMSO(vehicle control for curcumin) were studied for evidence of apoptosis by staining with viable dyes MC540 and Hoechst33342 and subsequently analyzed in the cell sorter. Results: Cur caused a concentration dependent inhibition of T cell proliferation in PMA-CD28 group(from 32775±3084 to 66±42 at 5.0 μg /ml of cur) and PHA group (from 50956±5747 to 24±12 at 5.0 μg/ml) with a calculated ED50 of 3.5 μM and 7.7μM respectively. CyA inhibited T cell proliferation in PHA group only with a calculated ED50 of 2.7 ng/ml but failed to block PMA + anti-CD28 stimulated T cell proliferation even at 250 ng/ml. In the apoptosis assay, T-cells from the PMA-CD28-cur had a significant increase in staining for MC540 as compared to the vehicle control with 0.4% DMSO in PMA-CD28 set (46±1% and 27±2% respectively, p<0.05) Conclusion: 1. Cur blocks the CyA resistant CD28 costimulatory pathway of T cell proliferation and therefore it may have novel adjuvant immunosuppressive properties. 2. Increased apoptosis may partially be responsible for this effect.

Monitoring early cellular responses in apoptosis is aided by the mitochondrial membrane protein-specific monoclonal antibody APO2.7

A recently described mitochondrial membrane protein-specific monoclonal antibody, APO2.7, was examined for monitoring early apoptotic responses in anti-CD95 (7C11)-induced Jurkat cells. Jurkat cells were harvested at 1.5, 3, 4.5, 6, 12, and 18 h after induction of apoptosis, and APO2.7 antibody monitored in unprocessed (no permeabilization agent used prior to staining) and processed (permeabilized prior to staining) cells. Light-scatter changes (decreased forward-scatter and increased side-scatter) by flow cytometry were observed after 3 h, and detection of cell permeability in unprocessed cells, as measured by light microscopic examination of Trypan blue-stained cells and flow cytometric detection of tubulin, showed little change until after 6 h. In addition, unprocessed cells stained with APO2.7 antibody showed little increase in staining until after 6 h following induction of apoptosis, when DNA fragmentation was demonstrated by flow cytometry and gel electrophoresis; however, processed cells stained with APO2.7 antibody showed significant increase in staining after 1.5 h. Detection, using annexin V and flow cytometry, of phospholipid membrane asymmetry from exposure of phosphatidylserine showed greater, apparent nonspecific staining in noninduced cells as compared to the other markers of apoptosis, but nearly paralleled the results of APO2.7 staining in processed cells from 3-18 h following CD95 induction of apoptosis. The data presented herein indicate that the mitochondrial membrane protein-specific antibody, APO2.7, is useful as a marker for the detection of apoptotic cells.

Visualization of the Distribution of Autophosphorylated Calcium/Calmodulin-Dependent Protein Kinase II after Tetanic Stimulation in the CA1 Area of the Hippocampus

Autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) at threonine-286 produces Ca2+-independent kinase activity and has been proposed to be involved in induction of long-term potentiation by tetanic stimulation in the hippocampus. We have used an immunocytochemical method to visualize and quantify the pattern of autophosphorylation of CaMKII in hippocampal slices after tetanization of the Schaffer collateral pathway. Thirty minutes after tetanic stimulation, autophosphorylated CaM kinase II (P-CaMKII) is significantly increased in area CA1 both in apical dendrites and in pyramidal cell somas. In apical dendrites, this increase is accompanied by an equally significant increase in staining for nonphosphorylated CaM kinase II. Thus, the increase in P-CaMKII appears to be secondary to an increase in the total amount of CaMKII. In neuronal somas, however, the increase in P-CaMKII is not accompanied by an increase in the total amount of CaMKII. We suggest that tetanic stimulation of the Schaffer collateral pathway may induce new synthesis of CaMKII molecules in the apical dendrites, which contain mRNA encoding its alpha-subunit. In neuronal somas, however, tetanic stimulation appears to result in long-lasting increases in P-CaMKII independent of an increase in the total amount of CaMKII. Our findings are consistent with a role for autophosphorylation of CaMKII in the induction and/or maintenance of long-term potentiation, but they indicate that the effects of tetanus on the kinase and its activity are not confined to synapses and may involve induction of new synthesis of kinase in dendrites as well as increases in the level of autophosphorylated kinase.