SPARCS and Pelli-Robson contrast sensitivity testing in normal controls and patients with cataract.

PurposeTo determine the ability of the newly developed internet-based Spaeth/Richman Contrast Sensitivity (SPARCS) test to assess contrast sensitivity centrally and peripherally in cataract subjects and controls, in comparison with the Pelli-Robson (PR) test.MethodsIn this prospective cross-sectional study, cataract subjects and age-matched normal controls were evaluated using the SPARCS and PR tests. Contrast sensitivity testing was performed in each eye twice in a standardized testing environment in randomized order. SPARCS scores were obtained for central, right upper (RUQ), right lower (RLQ), left upper (LUQ), and left lower quadrants (LLQ). PR scores were obtained for central contrast sensitivity. PR and SPARCS scores in cataract subjects were compared with controls. Intraclass correlation coefficients (ICC) and Bland Altman analysis were used to determine test-retest reliability and correlation.ResultsA total of 162 eyes from 84 subjects were analyzed: 43 eyes from 23 cataract subjects, and 119 eyes from 61 controls. The mean scores for SPARCS centrally were 13.4 and 14.5 in the cataract and control groups, respectively (P=0.001). PR mean scores were 1.31 and 1.45 in cataract and control groups, respectively (P<0.001). ICC values for test-retest reliability for cataract subjects were 0.75 for PR and 0.61 for the SPARCS total. There was acceptable agreement between the ability of PR and SPARCS to detect the effect of cataract on central contrast sensitivity.ConclusionsBoth SPARCS and PR demonstrate a significant influence of cataract on contrast sensitivity. SPARCS offers the advantage of determining contrast sensitivity peripherally and centrally, without being influenced by literacy.

Painless hypoglossal palsy as an isolated symptom of spontaneous carotid dissection.

Spontaneous internal carotid artery dissection (sICAD) occurs annually in 2.5 to 3 per 100,000 presenting with signs of ischemic events in the majority of cases. In contrast, lower cranial nerve palsy due to peripheral nerve affection is seldom the presenting clinical sign. In symptomatic cases (>90%), sICAD is most commonly accompanied by local pain. We report a case of a 49-year old woman with a left sICAD presenting with isolated ipsilateral hypoglossal palsy as the sole clinical sign. Compared to other cases, local pain was absent and other cranial nerves were not affected. Further, sICAD could not be detected in repeated Doppler-/Duplex-sonography, but magnetic resonance imaging and MR-angiography only.

Neurite transection produces cytosolic oxidation, which enhances plasmalemmal repair.

To survive, cells must rapidly repair (seal) plasmalemmal damage. Cytosolic oxidation has been shown to increase cell survival in some cases and produce cell death in other protocols. An antioxidant (melatonin; Mel) has been reported to decrease the probability of sealing plasmalemmal damage. Here we report that plasmalemmal damage produces cytosolic oxidation, as assayed by methylene blue (MB) color change in rat B104 hippocampal cells. Plasmalemmal sealing is affected by duration of Ca²âº deprivation and length of exposure to, and concentration of, oxidizing agents such as H2O2 and thimerosal (TH). Cytosolic oxidation by 10 µM to 50 mM H2O2 or 100 µM to 2 mM TH increases the probability of Ca²âº-dependent plasmalemmal sealing, whereas higher concentrations of H2O2 decrease sealing probability and also damage uninjured cells. We also show that antioxidants (Mel, MB) or reducing agents (dithiothreitol) decrease sealing. Proteins, such as protein kinase A, SNAP-25, synaptobrevin, and N-ethylmaleimide-sensitive factor (previously reported to enhance sealing in other pathways), also enhance sealing in this oxidation pathway. In brief, our data show that plasmalemmal damage produces cytosolic oxidation that increases the probability of plasmalemmal sealing, which is strongly correlated with cell survival in other studies. Our results may provide new insights into the etiology and treatment of oxidation-dependent neurodegenerative disorders, such as Parkinson's, Huntington's, and Alzheimer's diseases.

Reduction of nontarget infection and systemic toxicity by targeted delivery of conditionally replicating viruses transported in mesenchymal stem cells.

The fiber-modified adenoviral vector Delta-24-RGD (D24RGD) offers vast therapeutic potential. Direct injection of D24RGD has been used to successfully target ovarian tumors in mice. However, systemic toxicity, especially in the liver, profoundly limits the efficacy of direct viral vector delivery. Mesenchymal stem cells (MSC) have the ability to function as a vector for targeted gene therapy because of their preferential engraftment into solid tumors and participation in tumor stroma formation. We show that MSC-guided delivery of D24RGD is specific and efficient and reduces the overall systemic toxicity in mice to negligible levels compared with D24RGD alone. In our model, we found efficient targeted delivery of MSC-D24RGD to both breast and ovarian cell lines. Furthermore, immunohistochemical staining for adenoviral hexon protein confirmed negligible levels of systemic toxicity in mice that were administered MSC-D24RGD compared with those that were administered D24RGD. These data suggest that delivery of D24RGD through MSC not only increases the targeted delivery efficiency, but also reduces the systemic exposure of the virus, thereby reducing overall systemic toxicity to the host and ultimately enhancing its value as an anti-tumor therapeutic candidate.

The (in) auspicious role of mesenchymal stromal cells in cancer: be it friend or foe.

Recent progress in the research of mesenchymal stromal cells/multipotent stromal cells (MSC) has revealed numerous beneficial innate characteristics, suggesting potential value in an array of cellular therapies. MSC are easily isolated from bone marrow (BM), fat and other tissues, and are readily propagated in vitro. Transplanted/injected MSC have been shown to migrate to a variety of organs and tissues; however, sites of inflammation and pathology elicit enhanced MSC homing for tissue remodeling and repair. Tumors utilize many of the same inflammatory mediators uncovered in wound healing and likewise provide a site for preferential MSC homing. Although incorporation into the tumor microenvironment is apparent, the role of recruited MSC in the tumor microenvironment remains unclear. Some published studies have shown enhancement of tumor growth and development, perhaps through immunomodulatory and pro-angiogenic properties, while others have shown no apparent effect or have demonstrated inhibition of tumor growth and extended survival. This controversy remains at the forefront as clinical applications of MSC commence in anti-tumor therapies as well as as adjuncts to stem cell transplantation and in ameliorating graft-versus-host disease. Careful analysis of past studies and thoughtful design of future experiments will help to resolve the discrepancies in the field and lead to clinical utility of MSC in disease treatment. This review highlights the current theories of the role of MSC in tumors and explores current controversies.

Inflammation and tumor microenvironments: defining the migratory itinerary of mesenchymal stem cells.

Mesenchymal stem cells (MSC) exhibit tropism for sites of tissue damage as well as the tumor microenvironment. Many of the same inflammatory mediators that are secreted by wounds are found in the tumor microenvironment and are thought to be involved in attracting MSC to these sites. Cell migration is dependent on a multitude of signals ranging from growth factors to chemokines secreted by injured cells and/or respondent immune cells. MSC are likely to have chemotactic properties similar to other immune cells that respond to injury and sites of inflammation. Thus, the well-described model of leukocyte migration can serve as a reasonable example to facilitate the identification of factors involved in MSC migration. Understanding the factors involved in regulating MSC migration to tumors is essential to ultimately develop novel clinical strategies aimed at using MSC as vehicles to deliver antitumor proteins or suppress MSC migration to reduce tumor growth. For example, radiation enhances inflammatory signaling in the tumor microenvironment and may be used to potentiate site-specific MSC migration. Alternatively, restricting the migration of the MSC to the tumor microenvironment may prevent competent tumor-stroma formation, thereby hindering the growth of the tumor. In this review, we will discuss the role of inflammatory signaling in attracting MSC to tumors.

Principles of ultrasound imaging.

Ultrasound imaging is a process involving the real-time interaction of the physician with both the imaging equipment and the tissue. A good understanding of the physical principles involved in creating the image will ensure optimal handling of the equipment and proper interpretation of the resulting image.

A judge's three worlds: proof, philosophy, and the prison.

Tensions between the world of science and the world of law may arise because of their differing viewpoints and philosophies. Disagreements may center around such questions as what constitutes proof, around human behavior, and around the use of the insanity defense in criminal cases. The just deserts model is examined and is criticized as being harsh and possibly unrealistic in today's society.

Characterization of a T cell-derived lymphokine that acts synergistically with IL 3 on the growth of murine mast cells and is identical with IL 4.

A mast cell-like cell line (SN-1) was established with the aid of growth factor(s) present in the supernatant of a Con A-stimulated L3T4+ T cell line. In analogy to other mast cell lines, IL 3 was identified as a growth factor for SN-1 cells. In addition, a second lymphokine produced by the T cells synergistically enhanced the IL 3-induced growth. This factor, originally termed mast cell growth enhancing factor (MaGEF), could be separated from IL 2, IL 3, and a CSF-like activity and was purified to homogeneity. The N-terminal amino acid sequence (8 residues) and the functional properties of this lymphokine proved to be identical with those reported for BSF-1 (IL 4). Unless applied at high concentrations, purified MaGEF did not stimulate growth of the SN-1 mast cells in the absence of IL 3. MaGEF was also found to act on two IL 2-dependent T cell lines by inducing significant thymidine incorporation which was suboptimal compared to that induced by IL 2 and which cannot be inhibited by anti-IL 2-antibodies. A panel of cell lines developed from mouse bone marrow with IL 3 or with a combination of IL 3 and MaGEF all reacted to MaGEF in the presence of IL 3 with considerably increased proliferation. It is therefore suggested that one of the physiological functions of MaGEF is to promote the recruitment of T-dependent mast cells.

Butyrate-synchronized cloned T cells retain their dependence on interleukin-2 for growth induction. A model system for growth regulation.

Treatment of ST2/K9 cells, a cloned mouse T-cell line, with 1 mM sodium butyrate for 24 h leads to complete growth arrest in G1. This block is completely reversible and restimulation of cellular growth is entirely dependent on the presence of interleukin-2 (Il-2) in the culture medium. Additional as yet undefined serum factors are necessary for maintenance of further proliferation. After release from butyrate-induced growth arrest, Il-2 is required only during the induction phase of DNA replication. At the onset of thymidine incorporation, the growth factor can be removed, after which DNA replication occurs and the cells are able to complete only one cycle of duplication. The data presented here show that synchronization with sodium butyrate promotes cellular accumulation in the lymphokine-sensitive phase of the cell cycle. On the basis of the parameters established for restimulation of these cells, the detailed characterization of the molecular events involved in Il-2-mediated growth is possible.

Development of T cell clones reactive to two defined restriction elements in conjunction with two defined epitopes of antigen.

A previously described pig insulin (PI)-specific T cell line of (B10 X B10.BR)F1 origin was assayed for its reactivity with species variants of insulin in the presence of antigen-presenting cells (APC) of various H-2 haplotypes. In addition to its reactivity with PI and bovine insulin (BI) in the context of syngeneic F1 (H-2b X k)-APC, a weak cross-reactivity was observed with parental B10 (H-2b)-APC and BI but not PI. The cross-reactive cells could be selected out by several restimulations with the combination of BI and B10-APC. From the resulting, strongly cross-reactive T cell line several interleukin 2-dependent sublines were developed which did not require antigen-specific restimulations for further propagation. All such sublines had retained the original cross-reactivity with BI and B10-APC but showed significant differences in their fine specificity patterns, which indicates that each subline represents a clonal population. One of the sublines was cloned by limiting dilution at one cell/culture with a cloning efficiency of 76%. Five of the clones that were tested for reactivity had the same cross-reactivity as the original subline and upon recloning at 0.1 cells/culture the pattern again remained unchanged. From an analysis of the two antigen combinations (PI/F1 and BI/B10) it can be concluded that single cells can react with different restriction elements in the context of distinct epitopes of insulin. The implications of this finding for the mechanism of T cell recognition are discussed and a model for the function of major histocompatibility complex molecules in T cell recognition is proposed.

Bone marrow macrophages induced to antigen presentation by lymphokines interact selectively with distinct T cells.

In vitro matured bone marrow-derived macrophages (BMM phi), which represent a pure population of M phi, were shown to act as antigen-presenting cells (APC) to the T cell clone ST2/K.9. This interaction was major histocompatibility complex restricted. Upon long-term culture in macrophage colony-stimulating factor, BMM phi were activated for antigen presentation by a 48-h pulse with lymphokine-containing supernatant of concanavalin A-stimulated rat spleen cells (Con A sup). The capacity of such activated M phi to function as APC decreased upon removal of Con A sup, and could be regenerated by a second pulse. This finding suggests that antigen presentation by mature M phi is a reversible function regulated by T cell factors. When the responsiveness of various T cell lines to antigen presented on BMM phi or spleen cells was compared, distinct activation requirements were observed for different T cells since lymphokine-activated BMM phi were not capable of inducing antigen-specific proliferation of all lines.

Role of C3b receptors in the enhancement of interleukin-2-dependent T-cell proliferation.

The mechanism by which the complement system influences immune responses to T-cell-dependent antigens has not yet been clarified. That is why we studied the effect of the third complement component (C3) on different T-cell-dependent processes using well-defined mouse T-cell lines. While C3 did not influence the interleukin-2 (IL-2) production of the ST2/K-9 helper T-cells, the IL-2-dependent proliferation of the ST1 line was shown to be dose-dependently enhanced by C3. It is proved that neither the haemolytic activity of C3 nor the C3a fragment had any role in the process. The effect of C3 on the IL-2-dependent T-cell growth is even more enhanced (up to five-fold) when using polymerised C3. When the ST1 cell line is cultured in the presence of the cross-linked ligand, T-cells formed 80% less rosettes with red blood cells coated with antibody and mouse or human C3b. It is strongly suggested that C3--particularly when aggregated--exerts its enhancing effect on the growth of IL-2-dependent cell lines by binding to C3b receptors present on such T-cells.

Characterization of lymphokine-mediated activation of macrophages for antigen presentation: studies with long-term cultured bone marrow-derived macrophages and cloned T cells.

In cultures of bone marrow (BM) supplemented with L cell-derived colony-stimulating factor a pure population of macrophages (M phi) differentiates, which can be further propagated with a doubling time of 3.8 days. "Young" BMM phi obtained on day 8 of culture were shown to act as antigen-presenting cells inducing the antigen-specific proliferation of the cloned T cell line ST2/K.9, whereas "old" M phi had lost this ability. However, at any time tested (up to 132 days) the presentation function of old BMM phi could be completely restored by pulsing the cells with lymphokines (LK). A duration of 11 hr for the LK-pulse was sufficient to trigger the M phi to exert an optimal presentation function. This activity could be maintained when the LK-treatment was prolonged (tested up to 17 days). Activation was accompanied by a deceleration of growth. The LK effective in M phi activation were found to be contained in the supernatants of T cell lines stimulated by antigen or mitogen, and could be substituted by a low dose (5-10 units/ml) of recombinant interferon-gamma. In direct comparison LK-triggered BMM phi presented antigen as efficiently as peritoneal exudate M phi activated in vivo by ConA. Moreover, primed lymph node T cells responded to antigen-presenting BMM phi in a similar way as ST2/K.9 T cells. Therefore, these findings obtained with long-term cultured cells can be expected to reflect a physiological mechanism for the amplification of the immune response.

Epitope specificity and Ia restriction of T cell responses to insulin in a system of complementing Ir genes: analysis with primed lymph node T cells and a long-term cultured T cell line.

The antibody response of (H-2b X H-2k)F1 mice to pig insulin (PI) has previously been shown to be under the control of H-2-linked, complementing Ir genes. In addition, this response was reported to depend on the genetic background of the parental strains (Keck, K., Eur. J. Immunol. 1977. 7: 811). Here it is demonstrated that the secondary in vitro response of proliferating T cells shows the same dependence on H-2-linked Ir genes yet an influence of the background genes could not be detected. The complementing genes were mapped to the Kb, I-Ab and Kk, I-Ak regions. For restimulation of F1 T cells by PI, the Ir genes of both parental chromosomes have to be expressed in the same antigen-presenting cell, suggesting complementation at the molecular rather than at a cell interaction level. With a long-term cultured, PI-specific T cell line (ST2) of (B10 X B10.BR)F1 origin the complementation data could be confirmed by mapping the Ia restriction elements to Kb, I-Ab and I-Ak. The reactivity pattern of this line towards species variants of insulin and the isolated A and B polypeptide chains in the presence of syngeneic accessory cells suggests that the glutamic acid residue in position 4 of the A polypeptide chain (Asp in mouse insulin) is essential for recognition in conjunction with an (I-Ab X I-Ak)F1 hybrid Ia complex. I-Ab-encoded molecules carrying specificity Ia. W39 which, according to Rosenwasser, L. J. and Huber, B. T. are essential for the presentation of BI to (CBA/N X C57BL/6)F1 T cells, are not required as components of the F1-unique restriction element recognized by the F1 T cells of the ST2 line in conjunction with PI. This is indicated by the fact that accessory cells of (CBA/N X B10)F1 hybrids, regardless of their sex, could present PI as well as beef, sheep and horse insulin to the F1-restricted ST2 cells.

Induction of anamnestic T cell proliferation by antigen-pulsed, bone marrow-derived macrophages.

Bone marrow-derived macrophages (BMM phi) were grown in a liquid culture system in the presence of L cell-conditioned medium as a source of colony-stimulating factor. After a 4-h pulse with antigen, cultured irradiated BMM phi were capable of presenting the antigen to primed T cells as assessed in a T cell proliferation assay. Proliferation was optimal when BMM phi were used between days 5 and 8 of bone marrow cell culture. T cells of Lyt1 and Lyt123 phenotype had to be present at the start of the culture period to yield an optimal response. Conventional antisera and monoclonal antibodies directed against the H-2 I region and the I-A subregion, respectively, proved inhibitory in this system. Cultured BMM phi from low-responder strains failed to present antigens under immune response gene control in a form that was immunogenic to T lymphocytes. Cultured BMM phi might thus serve as a source of antigen-presenting cells in the study of cell-cell interaction and immune response gene regulatory mechanisms.

Messenger RNA of the large subunit of ribulose-1,5-bisphosphate carboxylase from Chlamydomonas reinhardi. Isolation and properties.

Polysomes specifically synthesizing the large subunit of ribulose-1,5-bisphosphate carboxylase were isolated from Chlamydomonas reinhardi cells by the indirect immunoprecipitation method. Electrophoretic analysis showed that the immunoprecipitated polysomes were of chloroplast origin. The mRNA coding for the large subunit which was purified from immunoprecipitated polysomes migrated at the 19-S position on sucrose density gradients, and its molecular weight was estimated to be 7.3 x 10(5) by acid-urea/agarose gel electrophoresis. The mRNA was translated in vivo with a cell-free protein-synthesizing system derived from Escherichia coli to give full-length large-subunit polypeptides.

The influence of suction catheter tip design on tracheobronchial trauma and fluid aspiration efficiency.

The suctioning efficiency and trauma-producing characteristics of five commercially available tracheobronchial suction catheters (Pharmaseal Tri-Flo, NCC Gentle-Flo, Argyle Aero-Flo, Argyle Dual Side-Hole, and Pharmaseal Whistle-Tip) were experimentally evaluated in anesthetized healthy dogs. The tendency of catheters to invaginate or "grab" tracheobronchial mucosa was observed with a bronchofiberscope during suctioning. Mucosal grabbing was seldom seen even at high (greater than 300 torr) vacuum levels with the cateter tip in the trachea. All catheters were observed to invaginate mucosa in lobar and segmental bronchi, with the frequency of grabbing being a function of airway anatomy, airway size, catheter orientation, tip design, and vacuum level. Catheters with multiple side-holes appeared to invaginate mucosa less frequently than the single side-hole catheter. Repeated suctioning of anesthetized healthy dogs followed by tracheobronchial excision, gross observation, and histologic examination of mucosal tissue biopsies demonstrated significant differences in the frequency and severity of lesions caused by the tracheobronchial suction procedure. All catheters were observed to damage airway lining, the damage related to multiple side-hole catheters appearing to be associated entirely with the act of cateter insertion and not with the application of vacuum. Only the Whistle-Tip design produced measurable damage beyond that related to catheter insertion. The average tip-suctioning effectiveness for each catheter, determined in vitro by aspirating a thin, uniform layer of simulated mucus, was found to be significantly higher for the Tri-Flo and Whistle-Tip catheters than the others, the Aero-Flo being least effective. Preliminary attempts to demonstrate this difference in suctioning effectiveness by comparing the performance of the catheters which displayed the highest and lowest tip suction effectiveness in a standardized clinical suctioning procedure revealed no significant difference in the percentage of mucus removed by either catheter. Additional studies should clarify this apparent contradiction.