Assessment of cell viability.

Cell viability may be judged by morphological changes or by changes in membrane permeability and/or physiological state inferred from the exclusion of certain dyes of the uptake and retention of others. This unit presents methods based on dye exclusion, esterase activity, and mitochondrial membrane potential, as well as protocols for determining the pre-fixation viability of fixed cells either before or after fixation. A dye-exclusion procedure for microscopy is also included.

Concentration of RB protein in nucleus vs. cytoplasm is stable as phosphorylation of RB changes during the cell cycle and differentiation.

Unphosphorylated RB (retinoblastoma tumor suppressor) protein is known to bind isolated nuclear matrix in vitro, whereas phosphorylated RB has a lower affinity, suggesting a mechanism which might contribute to differential nuclear/cytoplasmic localization as part of its regulatory activity. This motivates interest in the in vivo localization of the endogenous RB protein as its phosphorylation state changes during the cell cycle and cell differentiation. It is known that in proliferating HL-60 cells all the RB protein is phosphorylated, but the extent of phosphorylation increases with progression from G1 to S to G2 + M. It has also been previously shown that retinoic acid and 1,25-dihydroxy vitamin D3 shift the RB protein to the unphosphorylated state with cell differentiation (Yen, A., S. Varvayanis, Exp. Cell Res. 214, 250-257 (1994)). The dependence of cell cycle progression and differentiation on RB nuclear versus cytoplasmic localization, as well as the dependence of RB localization on phosphorylation state can thus be tested. Confocal image analysis of the RB protein in vivo shows that the ratio of the concentration of the RB tumor suppressor gene protein in the nucleus versus the cytoplasm remains stable as the RB protein undergoes either phosphorylation during cell cycle progression or dephosphorylation during cell differentiation induced by retinoic acid or 1,25-dihydroxy vitamin D3. For the cell cycle analysis, HL-60 human promyelocytic leukemia cells were fluorescently stained for DNA and for the RB protein. G1, S, and G2 + M subpopulations were isolated by fluorescence-activated cell sorting. For each subpopulation, the relative concentration of RB protein in the nucleus and the cytoplasm was measured by laser confocal image analysis. To determine the effect of retinoic acid-induced myeloid differentiation or 1,25-dihydroxy vitamin D3-induced monocytic differentiation, the same cell sorting and image analysis was performed on cells treated with these inducers. In all cases the concentration of the RB protein in the nucleus was approximately 2 times that in the cytoplasm. Thus, the ratio of nuclear versus cytoplasmic RB protein concentration is stable and independent of phosphorylation or dephosphorylation of RB during both the cell cycle and cell differentiation.

The major surface glycoprotein of Trypanosoma cruzi amastigotes are ligands of the human serum mannose-binding protein.

Trypanosoma cruzi, an obligate intracellular protozoan parasite, chronically infects mammals and causes Chagas' disease in humans. T. cruzi evasion of the mammalian immune response and establishment of chronic infection are poorly understood. During T. cruzi infection, amastigotes and trypomastigotes disseminate in the mammalian host and invade multiple cell types. Parasite surface carbohydrates and mammalian lectins have been implicated in the invasion of mammalian cells. A recent study has demonstrated that the human mannose-binding protein and the macrophage mannose receptor, two mammalian C-type lectins, bind to T. cruzi (S. J. Kahn, M. Wleklinski, A. Aruffo, A. Farr, D. Coder, and M. Kahn, J. Exp. Med. 182:1243-1258,1995). In this report we identify the major surface glycoproteins, including the SA85-1 glycoproteins, as T. cruzi ligands of the mannose-binding protein. Further characterization of the interaction between the mannose-binding protein and T. cruzi demonstrates that (i) the SA85-1 glycoproteins are expressed by amastigotes and trypomastigotes but only amastigotes express the mannose-binding protein ligand, (ii) treatment of amastigotes with alpha-mannosidase inhibits the binding of mannose-binding protein, and (iii) amastigote binding of mannose-binding protein is stable despite the spontaneous shedding of some glycoproteins from its surface. Together, the data indicate that developmentally regulated glycosylation of surface glycoproteins controls the expression of ligands that affect the interactions between T. cruzi and mannose-binding protein. It has been established that the binding of mannose-binding protein to microorganisms facilitates their uptake into phagocytic cells. Preferential opsonization of amastigotes with mannose-binding proteins may account for their clearance from the circulation and may contribute to the parasite's ability to invade different cell types.

Trypanosoma cruzi amastigote adhesion to macrophages is facilitated by the mannose receptor.

Trypanosoma cruzi is an obligate intracellular protozoan parasite. The mammalian stage of the parasite life cycle describes amastigotes as an intracellular form that replicates, and trypomastigotes as an extracellular form that disseminates and invades cells. Recent studies, however, have demonstrated that amastigotes circulate in the blood of infected mammals and can invade mammalian cells. In this report, a T. cruzi surface glycoprotein gene, SA85-1.1, was expressed as an immunoglobulin chimera, and this recombinant globulin was used to screen normal mouse tissues for adhesive interactions. This approach identified a subset of macrophages in the skin and peripheral lymph node that bind the T. cruzi surface glycoproteins through the mannose receptor. To further examine the T. cruzi mannose receptor carbohydrate ligands, the interaction between T. cruzi and the mannose-binding protein, a mammalian lectin with similar carbohydrate binding specificities as the mannose receptor, was examined. These studies demonstrated that the mannose-binding protein recognized amastigotes, but not trypomastigotes or epimastigotes, and suggested that amastigotes would also be recognized by the mannose receptor. Therefore, amastigote adhesion to macrophages was investigated, and these experiments demonstrated that the mannose receptor contributes to amastigote adhesion. The data identify the first mammalian lectins that bind to T. cruzi, and are involved in T. cruzi invasion of mammalian cells. The data suggest that amastigotes and trypomastigotes may have developed different mechanisms to adhere to and invade host cells. In addition, it has been established that IFN-gamma-activated macrophages express low levels of the mannose receptor and are trypanocidal; this suggests that the interaction between amastigotes and the mannose receptor enables amastigotes to increase their adherence with a population of macrophages that are nontrypanocidal and permissive for their intracellular replication.

Computing the central location of immunofluorescence distributions: logarithmic data transformations are not always appropriate.

The idea of the "average" intensity of immunofluorescence data is often poorly defined, with such terms as average, mean, and peak used interchangeably. In addition, the common use of logarithmic amplifiers with immunofluorescence data further complicates the problem. Log amplifiers permit the display of a wider range of fluorescence intensities. At the same time, they effect a log transformation of the data. This transformation decreases the variance resulting in narrower fluorescence distributions, which are assumed to approximate normal distributions. When the log transform is used, the distribution mean is the geometric mean of the untransformed data, which is computed simply as the mean of the channel values. This mean value serves as a simple indicator of the population center. Despite the prevalence of log transformations in flow cytometry, this transformation may not yield normally distributed immunofluorescence data, whereas the square root or other fractional power transformations can yield normal distributions.

Cell subset (CS) parameter to record the identities of individual cells in flow cytometric data.

The Flow Cytometry Standard (FCS) for cytometric data (Dean et al.: Cytometry 11:323-332, 1990) provides for appending an ANALYSIS section to a data file, but it does not explicitly provide for recording the identities of individual cells. We propose an extension to the FCS definition in order to record the identity of each individual cell in list mode data. In order to do so, one first defines the subpopulations of cells to be identified and one than assigns a number to each defined population. For example, in an analysis in which peripheral blood mononuclear cells are labeled with antibodies to CD3, CD4, and CD8, the "negative" lymphocytes that are labeled with none of the antibodies could be identified as population 1, the CD3-CD4-CD8+ lymphocytes as population 2, etc. As the measured values from each cell are analyzed, the number that identifies the population to which that cell belongs is assigned as the value of an additional parameter. Since this procedure merely adds a new parameter, the only necessary extension to the FCS specification is that a particular name is recognized for this parameter. We propose that CS (abbreviation for cell subset) be recognized as the name and that CS be used as the value for the $PnN (parameter name) keyword in the FCS file TEXT. The CS values that are created can be used to aid in data analysis and can be permanently recorded in a conventional FCS file. A data file that is saved with CS values includes an explicit and integral record of the complete analysis, regardless of the complexity of the analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased intracellular calcium is associated with progression of HPV-18 immortalized human keratinocytes to tumorigenicity.

Studies were conducted using normal and human papillomavirus Type 18 (HPV-18) immortalized human keratinocytes to assess possible alterations in the differentiation process as a consequence of increased intracellular calcium concentration. Normal keratinocytes exposed to increased extracellular calcium or the phorbol ester TPA, exhibited terminal differentiation characteristics. However, late passage HPV-18 immortalized keratinocytes (designated FEP-1811) were resistant to such terminal differentiation signals. Flow cytometric analyses of 1811 cells at various stages of passage in culture revealed progressively higher levels of intracellular calcium in the immortalized cells with passage in culture when compared to normal, primary keratinocytes. Furthermore, 1811 cells isolated from tumors which developed in irradiated nude mice contained the highest level of intracellular calcium of all the cells examined. These results suggest that an increase in the concentration of intracellular calcium is associated with progression of HPV-18 immortalized keratinocytes to tumorigenicity.

Long-term effects of fermentable fibers on rat colonic pH and epithelial cell cycle.

The long-term effects of fermentable fibers on colonic luminal pH and the epithelial cell cycle were compared in 50 male Sprague-Dawley rats fed either a defined basal fiber-free diet or the basal diet supplemented with 10% pectin, cellulose or guar or with 20% oat bran. After 8 mo, in vivo pH measurements revealed that acidification of luminal contents occurred in the cecum and in mid and distal colons of rats fed fiber-supplemented diets when compared with the fiber-free controls (P less than 0.05). Pectin and guar produced the greatest acidification of luminal contents, the largest increase in cecal surface area and the highest percentage of colonic cells in S-phase, as measured by flow cytometry. In the proximal colon of the pectin group 9.2 +/- 0.5% of cells were in S-phase (6.3 +/- 0.8% with the fiber-free group) (P less than 0.05) and in the distal colon of the guar group 10.9 +/- 1.4% were in S-phase (7.1 +/- 0.5% with the fiber-free group) (P less than 0.05). Even though the most fermentable fibers produced the greatest mitogenic response, there was no site-specific correlation between pH and mucosal cell growth except in the cecum. This suggests that fibers may act as colon cell growth factors by some mechanism other than extracellular pH changes.

Influence of luminal pH on rat large bowel epithelial cell cycle.

The influence of luminal pH on the colonic epithelial cell cycle was studied by means of dietary modification to produce acidification of colonic contents. Forty male Sprague-Dawley rats were fed one of four diets: defined fiber free, fiber free diluted by either 100 g/kg lactulose or sorbitol, or 25 g/kg of MgSO4. After 2 wk, in vivo pH measurements were recorded at laparotomy under ether anesthesia in the cecum and proximal, mid-, and distal colon. Epithelial cells, isolated from these same regions, were measured for DNA content with flow cytometry in order to determine cell cycle stage. The pH values in all intestinal regions were highest with MgSO4 and then fiber free, sorbitol, and lactulose, the most acidic. In contrast, the percentage of cells in S phase (actively synthesizing DNA) was highest with lactulose and least with MgSO4. There was a significant inverse correlation between luminal pH and percent cells in S phase in the cecum (P less than 0.01), proximal colon (P less than 0.05), and distal colon (P less than 0.01). These results show that acidification of colonic contents by diet modification leads to increased epithelial cell proliferation.

Midgut volvulus following cesarean section.

Total midgut volvulus and gangrene due to a congenitally elongated mesentery in a 27-year-old primipara occurred 3 days after cesarean section. All of the small bowel distal to the duodenum except for 12 inches of jejunum was resected, and the patient survived. This case and the literature concerning volvulus in pregnancy are reviewed here, and suggestions for prevention and treatment are made.