Differences In Cell Populations Research Articles

Biofilm formation by Aeromonas hydrophila on green-leafy vegetables: cabbage and lettuce.

Aeromonas hydrophila is the most well known of the six species of Aeromonas, which has been linked to two groups of human diseases: septicemia and gastroenteritis. Reference strain ATCC 7966 and biofilm strains TUB19, TUB20, and TUB21 were investigated for their ability to form biofilm in vitro (after 48 h on polystyrene surface) and on the surface of two green-leafy vegetables, cabbage and lettuce (after 1, 2, 4, and 24 h). Attachment strength (S(R)) of these strains to the vegetable surface was also measured in the same time intervals. The ATCC 7966 and TUB19 had high ability to form biofilm in vitro compared with TUB20 and TUB21 in full strength tryptone soy broth or under starvation conditions in diluted tryptone soy broth (1:20, v/v). Cell surface hydrophobicity of the biofilm strains was lower than that of the reference strain. The biofilm of all tested strains on polystyrene surfaces differed from that on the vegetable surfaces. All strains studied rapidly attached to both green leafy vegetables (after 1 h). S(R) and cell populations (loosely and strongly attached cells) significantly (p < 0.05) increased with contact time; however, no significant (p > 0.05) differences in cell populations were recorded after 4 and 24 h. The highest S(R) and cell population (log CFU cm⁻²) were recorded by TUB19. In conclusion, the use of A. hydrophila strains isolated from environmental biofilm samples may be more useful for understanding biofilm formation on green-leafy vegetables than the reference or laboratory strains. The attachment of A. hydrophila was significantly affected by the surfaces of green-leafy vegetables. Further studies are required to improve our understanding of the interaction between human microbial pathogens and surfaces of raw vegetables.

Regionalization of pIgR expression in the mucosa of mouse small intestine

Few reports exist on the differences in cell populations or immunological functions between the proximal and distal segments of the small intestine (SI). In the current contribution we analyzed the expression of the polymeric immunoglobulin receptor (pIgR) and alpha chains as well as the density of IgA-producing cells from the proximal and distal intestinal segments from Balb/c mice. Furthermore, by using real-time RT-PCR we quantified the expression of cytokines (TNF-α, IFN-γ, IL-4 and TGF-β), Toll-like receptor-4 (TLR-4), and the glucocorticoid receptor (GR) involved in pIgR expression in intestinal epithelial cells (IEC). In this study, for the first time it has been demonstrated that the expression of the pIgR as well as alpha chain was greater in the proximal than the distal segment of the small intestine of normal mice. Moreover, we found striking differences in the expression of cytokines at the different intestinal compartments. Whereas the expression of TNF-α, IFN-γ and TGF-β was higher in lamina propria lymphocytes (LPL) of the distal than proximal segment, it was higher in IEC of the proximal than distal segment. In contrast, the expression of the gene for IL-4 was higher in the LPL of the proximal segment and the IEC of the distal segment. Although the overall expression of TNF-α, IL-4, IFN-γ and TGF-β was higher in the whole mucosa of the distal than proximal segment, we propose that cytokines produced by epithelial cells (TNF-α, IFN-γ and TGF-β) autocrinally up-regulate the expression of mRNA for the pIgR. Finally the expression of the GR was higher in the proximal segment, while the expression of the gene for TLR-4 was significantly higher in the IEC of the distal than proximal segment. The higher expression of pIgR found in the proximal segment is probably related to the effect on epithelial cells of the higher production of TNF-α, IFN-γ and TGF-β, as well as the higher expression of the glucocorticoid receptors. The increased expression of pIgR in the proximal segment appears primarily responsible for the increased secretory IgA levels in the small intestine of mice. These results confirm and extend previous findings supporting the compartmentalization of the intestinal immune system.

Are animal models useful for studying human disc disorders/degeneration?

Intervertebral disc (IVD) degeneration is an often investigated pathophysiological condition because of its implication in causing low back pain. As human material for such studies is difficult to obtain because of ethical and government regulatory restriction, animal tissue, organs and in vivo models have often been used for this purpose. However, there are many differences in cell population, tissue composition, disc and spine anatomy, development, physiology and mechanical properties, between animal species and human. Both naturally occurring and induced degenerative changes may differ significantly from those seen in humans. This paper reviews the many animal models developed for the study of IVD degeneration aetiopathogenesis and treatments thereof. In particular, the limitations and relevance of these models to the human condition are examined, and some general consensus guidelines are presented. Although animal models are invaluable to increase our understanding of disc biology, because of the differences between species, care must be taken when used to study human disc degeneration and much more effort is needed to facilitate research on human disc material.

Effects of Cord Blood Cell Subset Populations in the Development of the Dominant Cord Blood Unit in Non-Myeloablative Sequential Double Cord Blood Transplantation (DCBT).

Umbilical cord blood has become an established alternative hematopoietic stem cell source for patients without suitable donors. However, single cord blood transplantation in adults has been associated with high mortality rates due to graft failure, delayed engraftment, and poor immune reconstitution. In order to improve engraftment, sequential cord blood transplantation has been employed. One consistent observation with this strategy has been the eventual dominance of one cord graft over the other. However, the factors that determine the fate of one cord over the other have not been well elucidated. Our previous work demonstrated that the dominant cord tends to have an initial higher CD34+ cell dose. This study attempted to identify other cell populations in the cord units that may determine which unit becomes the ultimate source of hematopoiesis. Forty patients with hematologic malignancies underwent non-myeloablative conditioning with fludarabine, melphalan, and ATG followed by two sequential cord blood infusions. All cord units were at least a 4/6 HLA match with the recipient and each other. GVHD prophylaxis consisted of cyclosporine & mycophenolate mofetil or FK-506 & rapamycin. Chimerism analysis of peripheral blood leukocytes was performed by PCR of short tandem repeat loci. Flow cytometry for T-cells (CD4 and CD8), B-cells (CD19), and NK cells (CD3−CD56+CD16− & CD3−CD56+CD16+) were done using standard immunofluorescence methods. A total of 10 patients were available for this analysis. The Wilcoxon signed rank test was used to evaluate the significance of the difference in cell populations between the transplanted cords on graft outcome. This analysis demonstrated that 80% (8 of 10) of the dominant cords had higher NK cells when compared to the rejected unit with average total NK cells of 4.15 x 106 vs 2.30 x 106 per kg of recipient, respectively. Total NK cells included both CD16− and CD16+ cells, but the difference was much higher for CD16+ NK cells, which represent a more mature NK phenotype. B-cells, CD4+ T-cells, and CD8+ T-cells were similar between the two cords. The mean total cell/kg of recipient for each subpopulation between cord units are summarized in Table 1. A trend towards a difference in NK cells (p=0.1) was observed; the small number of samples, however, limited the interpretation of the analysis. Future studies involving additional DCBT recipients are ongoing to better define the influence of NK cells as well as other cell populations including mesenchymal stem cells and T-reg cells on cord graft outcome.Mean Subpopulation Cell Count/kg of recipient between Cord UnitsCordCD3+CD4+ T-CellsCD3+CD8+ T-CellsCD3−CD56+CD16− NK CellsCD3−CD56+CD16+ NK CellsTotal NK CellsCD19− B-CellsDominant Cells/kg3.92E+06 (1.35–7.30E+06)1.76E+06 (0.5–3.22E+06)4.64E+05 (0.06–1.52E+06)3.68E+06 (0.4–9.65E+06)4.15E+06 (0.7–9.97E+06)1.68E+06 (0.3–3.96E+06)Rejected Cells/kg4.34E+06 (2.04–7.83E+06)1.71E+06 (0.7–4.86E+06)3.60E+05 (0.0–0.9E+06)1.94E+06 (0.3–3.55E+06)2.30E+06 (0.3–4.10E+06)1.80E+06 (0.9–3.15E+06)

Matrices with Compliance Comparable to that of Brain Tissue Select Neuronal over Glial Growth in Mixed Cortical Cultures

Cortical neurons and astrocytes respond strongly to changes in matrix rigidity when cultured on flexible substrates. In this study, existing polyacrylamide gel polymerization methods were modified into a novel method for making substrates capable of engaging specific cell-adhesion receptors. Embryonic cortical dissociations were cultured on polyacrylamide or fibrin gel scaffolds of varying compliance. On soft gels, astrocytes do not spread and have disorganized F-actin compared to the cytoskeletons of astrocytes on hard surfaces. Neurons, however, extend long neurites and polymerize actin filaments on both soft and hard gels. Compared to tissue culture plastic or stiff gel substrates coated with laminin, on which astrocytes overgrow neurons in mixed cultures, laminin-coated soft gels encourage attachment and growth of neurons while suppressing astrocyte growth. The number of astrocytes on soft gels is lower than on hard even in the absence of mitotic inhibitors normally used to temper the astrocyte population. Dissociated embryonic rat cortices grown on flexible fibrin gels, a biomaterial with potential use as an implant material, display a similar mechano-dependent difference in cell population. The stiffness of materials required for optimal neuronal growth, characterized by an elastic modulus of several hundred Pa, is in the range measured for intact rat brain. Together, these data emphasize the potential importance of material substrate stiffness as a design feature in the next generation of biomaterials intended to promote neuronal regeneration across a lesion in the central nervous system while simultaneously minimizing the ingrowth of astrocytes into the lesion area.

The use of porcine small intestinal submucosa as a biomaterial for perineal herniorrhaphy in the dog.

To develop an in vivo perineal hernia model, to develop a technique for using small intestinal submucosa (SIS) in perineal hernia repair, to further elucidate the biological behavior of SIS, and to compare SIS herniorrhaphy with the internal obturator muscle transposition (IOT) technique. Prospective evaluation comparing SIS herniorrhaphy with IOT. Twelve adult castrated male, large-breed dogs. All dogs had bilateral pelvic diaphragm defects created by complete excision of the levator ani muscle. Each dog had one side repaired using SIS and the other by IOT. Pain and inflammation were subjectively scored. Dogs were killed 2 weeks (n = 4), 12 weeks (n = 4), or 16 weeks (n = 4) after surgery. Each pelvic diaphragm was biomechanically tested to failure. The pelvic diaphragms from 2 normal dogs (n = 4 sides) were also biomechanically tested. Failure site, maximum pressure, displacement at failure, and initial linear stiffness values were determined. Histologic assessment was performed. Statistical analysis was performed with significance set at P <.05 No significant postoperative complications were noted. There were no significant differences in maximum pressure to failure, displacement, or stiffness when comparing normal, SIS, and IOT at any time point. The SIS group had significantly less displacement (P =.004) at 2 weeks than at weeks 12 or 16. For all herniorrhaphy techniques, the failure site was central (n = 22) or at the suture line (n = 2). At 2 weeks, histologic evaluation of tissues from the IOT group showed inflammation, mineralization, and necrosis, which were not present in tissues from the SIS group. Histologic examination at 12 and 16 weeks showed no microscopic differences in cell population or tissue characteristics between the IOT and SIS groups. SIS herniorrhaphy was successfully performed in this in vivo model of perineal hernia in the dog. This study suggests that SIS can be used as a primary means of repair, as augmentation when the internal obturator muscle is thin and friable, or as a salvage procedure in cases of recurrence in dogs with perineal hernia.

The MDCK cell line is made up of populations of cells with diverse resistive and transport properties

The MDCK cell line has been used as a model for the renal distal tubule. When grown on permeable supports, the cells form tight junctions and transport sodium and calcium. Widely different values have, however, been obtained for resistance and transports by different investigators. Since the MDCK cell line is genetically heterogeneous, one potential reason for observed differences is the use of different populations of cells. Dilutional techniques have been used to obtain eleven MDCK cell clones. The clones exhibit a range of values for resistance (300–4000Ω cm2), net Ca2+transport (3.2–81pmol /cm2/min) and net Na+transport (0.4–6.0nmol/cm2/min). To determine if the characteristics of clonal cell lines changed with increasing passage number, resistance was measured at relatively early and late passage number. Resistance increased with increasing passage number in two of six lines and decreased in one. These studies demonstrate that differences between previous studies may be due to differences in cell population, length of time since passage, and passage number. Clonal cell lines should provide a useful tool to investigate ion transport and mechanisms of regulation of transport in this model of renal distal tubule.

Analysis of Rainbow Trout Peripheral Blood Leucocytes Separated by Flow Cytometry Cell Sorting.

The characterization of rainbow trout peripheral blood leucocytes (PBL) was carried out by flow cytometry (FCM) analysis and by transmission electron microscopy. Two major cell populations were identified in FCM analysis. One population showed small values in both forward scatter (FS) and side scatter (SS), and the other population showed large values in both parameters. These major populations were divided into two sub-regions each (A/B and C/D). There was no significant difference in cell populations between sub-regions A and B. However, a difference was observed in the small and large lymphocyte percentages between sub-regions C and D. The majority of regions (A and B) were constituted by neutrophils (56% and 66%, respectively), and monocytes accounted for 9% and 7% of the cells, respectively. Large and small lymphocytes were also present in these regions, accounting for 26% and 6% of the cells in sub-region A and 14% and 7% in sub-region B, respectively. In sub-regions C and D, cells were mainly constituted by large and small lymphocytes (33% and 49% in C, and 7% and 70% in D, respectively) and thrombocytes (14% and 23%).

Pale islands in medulloblastoma consist of differentiated cells with low growth potential.

Cerebellar medulloblastomas are the most common embryonal neoplasms occurring in the pediatric age group. Thirty-five specimens from 28 cases of medulloblastoma and variant neoplasms were examined to elucidate the histopathologic significance of the reticulin-free pale islands. In 10 specimens, there were light-looking areas corresponding to pale islands: three specimens from desmoplastic and seven from classic medulloblastoma. The difference in cell population, 1.73-fold more cells in dark areas than in light areas (P < 0.001), seemed to be a major factor in the formation of the biphasic architecture. Immunohistochemically, occasional tumor cells in both areas were found to express neuronal phenotypes. In addition, positive immunoreactivity for glial markers was exceptional. There was significant difference in the staining indices (SI) for cell proliferation markers. The light areas consistently showed low SI values (mean MIB-1 SI, 5.75; mean PCNA SI, 11.8) compared with the dark areas (mean MIB-1 SI, 22.7; mean PCNA SI, 44.9). It is suggested that light areas (pale islands) consist of more differentiated cells of neuronal lineage with relatively low proliferation potential. These tumor cells might proliferate in a group, and then expand, thereby compressing the surrounding tissue to form pale islands.

Topographic Differences in Cell Populations and Insulin Secretion in the Endocrine Pancreas of the ToadBufo arenarum

We analyzed the endocrine cell topography within the amphibian pancreas and the relationship of this distribution to lobular variation in insulin content and secretion. Pancreases from adult male toadBufo arenarumwere separated into their five lobes: free, gastric, hepatic, duodenal, and jejunal. Pieces of each lobe were incubated with glucose, arginine, and K+and the insulin concentration in the medium was measured by radioimmunoassay. In the presence of 2 or 8 mMglucose, 10 mMarginine, and 10 mMK+the free lobe released a significantly greater amount of insulin than the other lobes, while the output of the gastric lobe was greater than that of the duodenal, hepatic, and jejunal. At 8 mMglucose, every pancreatic lobe released a significantly higher amount of insulin than at 2 mM. The insulin content of the free lobe was significantly higher than that of the others, whereas this parameter was comparable among the latter. These pancreases contained islets of variable size and irregular shape. B and non-B cells, detected by immunoperoxidase staining, were located at the central and peripheral zones of the islets, respectively. A large number of non-B cells were also scattered over the exocrine component. Morphometrical analyses revealed the following sequence of endocrine cell percentage: free lobe > gastric lobe = duodenal lobe > jejunal lobe = hepatic lobe. Some 48% of the endocrine cells were present in the islets, while the remaining 52% were found throughout the exocrine pancreas. In the free lobe, more endocrine cells were located within the islets (65%) than outside and in the gastric lobe the proportion was almost equal (48% within, 52% outside), but in the hepatic, duodenal, and jejunal lobes the majority lay outside the islets (61, 63, and 70% extrainsular, respectively). The area covered by B and D cells was far larger within the islets than outside, with the relative magnitude of this difference being free lobe > gastric lobe > duodenal lobe > hepatic lobe = jejunal lobe. In the free lobe, this relative distribution was more skewed than in the remaining lobes. PP and A cells occupied a more extensive area outside the islets than inside in every lobe. There were no significant differences among the extrainsular areas occupied by each type of endocrine cell within a given pancreatic lobe. These results constitute the first demonstration of the heterogeneity in morphology, insulin content, and secretory function among the pancreatic lobes inB. arenarum.The data further suggest that the nonuniform secretory capacities of amphibian pancreatic lobes reflect localized differences in their insulin content, which heterogeneity in turn stems from the dissimilar distribution and organization of their constituent endocrine-cell populations.

Use of isografts to study proliferation and differentiation programs of mouse stomach epithelia

A new model system has been developed for studying the factors that regulate the proliferation and differentiation programs of the principal cell lineages present in the mouse stomach epithelium. When embryonic day 14 (E14) stomachs from C57Bl/6 mice are implanted under the subcutaneous fascia of young adult male CBY/B6 nude mouse recipients, they are rapidly vascularized and begin to grow at rates comparable to the intact stomach of a similarly aged animal. Single- and multilabel immunohistochemical studies, conducted in the intact stomach of E14 to postnatal day 42 (P42) C57Bl/6 mice and in P7, P14, P28, and P42 C57Bl/6 isografts, suggest that the apparently undifferentiated glandular epithelium of E14 stomach already has encoded positional information that permits it to establish regional differences in cell populations even in the absence of exposure to normal luminal contents. Regional differentiation is manifested by the establishment of distinct zones, each composed of gastric units with zone-specific cell lineages. This axial patterning evolves at a rate similar to that of the intact stomach, even though the E14 isograft is placed in the hormonal environment of an adult host and therefore cannot be programmed by normal developmental changes in endocrine status. Moreover, patterning is maintained in isografts despite continuous renewal of its epithelial cell lineages. Although isografts apparently possess autonomously functioning temporal and spatial programs, perturbations were noted in the proliferation and/or differentiation programs of certain lineages, e.g., the rate of accumulation of endocrine cell subpopulations is reduced in these grafts.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione S-transferase isoenzymes in rat brain neurons and glia

The glutathione S-transferases (GSTs) constitute a family of cytosolic isoenzymes and a structurally unrelated microsomal enzyme that is involved in the detoxication of electrophilic xenobiotics. These enzymes also participate in the intracellular binding and transport of a broad range of lipophilic compounds including bilirubin, and hormones such as the glucocorticoids and thyroid hormones. The present investigation demonstrates that GSTs are present in neurons of the brainstem, forebrain, and cerebellum. An isoenzyme-specific distribution of GSTs was found in cytoplasm, nuclei, and nucleoli. The regional and cellular distribution of cytosolic GSTs in the brain was studied by immunohistochemistry, spectrophotometric enzyme assay, and reverse-phase HPLC. Polyclonal antibody against microsomal GST was strongly reactive with Purkinje cells throughout the cerebellar cortex, and with neurons in the brainstem and hippocampus. Nuclei of Purkinje cells and of neurons in the brainstem, hippocampus, and cerebral cortex were immunopositive for alpha-class GST 1-1 (YaYa), whereas alpha-class GST 2-2 (YcYc) antibody was consistently immunoreactive with the nucleolus, but not with the nucleus or soma. All alpha-class GST antibodies studied were reactive, to various degrees, with astrocytes and choroid plexus; however, ependymal cells of the subventricular zones were immunonegative. alpha-class GST 8-8 (YkYk) immunoreactivity was specifically localized to endothelial cells and/or astrocytic end feet associated with blood vessels. Reverse-phase HPLC indicated that there were also substantial regional differences in the pattern of alpha-, mu-, and pi-class GST subunit expression. For example, the thalamus/hypothalamus had the highest GST activity and greatest concentration of total GST protein and mu-class GST subunit 6 (Yb3), whereas the brainstem had the greatest concentration of pi-class GST subunit (Yp). This regional variation in GST expression may be reflective of regional differences in cell populations. In cerebellar cortex, the concentration of mu-class GST subunit 4 (Yb2) was greatest in the flocculus and lowest in the vermis. This is of clinical interest because the pattern of expression of mu-class GST subunit 4 (Yb2) in the cerebellum coincides with the known regional susceptibility of this structure to degeneration after exposure to toxic or metabolic insults. The vermis is most susceptible to these insults, whereas the lateral lobes and flocculus are most resistant.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell populations and episodic periodontal attachment loss in humans*

The purpose of the present study was to assess possible associations between episodic probing attachment loss and cell populations in the supracrestal connective tissue in humans. 10 systemically healthy adult patients with untreated advanced periodontitis were monitored during a period of 10 months. At baseline and every month thereafter, probing attachment levels were measured at 6 sites of every tooth using an electronic pressure sensitive probe and flexible stents. Corresponding contralateral sites were identified where 1 site had lost 2 mm or more attachment within the previous month (P), and the other site had not (C). Supracrestal soft tissue biopsies were taken from these sites, processed and cut into 1 micron sections. Cell populations were identified in superficial and deep connective tissue areas by counting fibroblasts, macrophages, plasma cells, mast cells, granulocytes, lymphocytes, endothelial cells and the total number of inflammatory cells. Analysis of variance assessed differences in cell populations between P- and C-sites. There were statistically significantly higher numbers of fibroblasts in the standard areas of C-sites (p less than 0.0001). In P-sites, the numbers of macrophages, plasma cells, mast cells, lymphocytes and total inflammatory cells were significantly higher as compared to C-sites (p = 0.05-0.0001). There were no differences in cell populations between superficial and deep connective tissue areas within P- and C-sites (p greater than 0.2). Clinically assessed episodes of periodontal disease progression may be associated with site-specific shifts in inflammatory cell populations.

Recombinant human granulocyte colony-stimulating factor repairs the abnormalities of neutrophils in patients with myelodysplastic syndromes and chronic myelogenous leukemia

We examined the in vitro effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on neutrophil anomalies in 20 patients with myelodysplastic syndromes (MDS) and eight patients with chronic myelogenous leukemia (CML). Neutrophil alkaline phosphatase (NAP) activity was determined in nine MDS patients and eight CML patients by a scoring method. NAP scores were decreased in six of the nine patients with MDS and in all of the patients with CML. In all patients with these diseases, NAP scores increased by incubating the blood with rhG-CSF. An increase in NAP scores by rhG-CSF was observed even at a concentration of 1 U/mL in patients with MDS but was observed only at higher concentrations (1,000 to 10,000 U/mL) in patients with CML. Significant increases in NAP scores occurred at 12 hours' incubation in patients with MDS, whereas the increase was more gradual in patients with CML. This time course difference was thought to be due mainly to the difference in cell populations of circulating myeloid cells between MDS patients and CML patients. Induction of NAP activity by rhG-CSF in patients with both these diseases was suppressed by the addition of inhibitors of RNA or protein synthesis. Neutrophil superoxide anion (O2-) production induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was determined in the other 11 patients with MDS. This neutrophil function was decreased in seven of the 11 patients with MDS, normal in two patients, and increased in two patients. Preincubation with rhG-CSF caused a significant increase in fMLP-induced O2- production in nine of the 11 patients with MDS. rhG-CSF enhanced this neutrophil function in a time- and dose-dependent manner, and maximal stimulation was observed at 2,000 to 4,000 U/mL of rhG-CSF and at five to ten minutes' incubation. The present results show that rhG-CSF is able to repair at least in part the neutrophil anomalies in these patients, and our data, especially for patients with MDS, suggest the clinical usefulness of rhG-CSF for this preleukemic disorder.

Recombinant Human Granulocyte Colony-Stimulating Factor Repairs the Abnormalities of Neutrophils in Patients With Myelodysplastic Syndromes and Chronic Myelogenous Leukemia

Abstract We examined the in vitro effect of recombinant human granulocyte colony- stimulating factor (rhG-CSF) on neutrophil anomalies in 20 patients with myelodysplastic syndromes (MDS) and eight patients with chronic myelogenous leukemia (CML). Neutrophil alkaline phosphatase (NAP) activity was determined in nine MDS patients and eight CML patients by a scoring method. NAP scores were decreased in six of the nine patients with MDS and in all of the patients with CML. In all patients with these diseases, NAP scores increased by incubating the blood with rhG- CSF. An increase in NAP scores by rhG-CSF was observed even at a concentration of 1 U/mL in patients with MDS but was observed only at higher concentrations (1,000 to 10,000 U/mL) in patients with CML. Significant increases in NAP scores occurred at 12 hours' incubation in patients with MDS, whereas the increase was more gradual in patients with CML. This time course difference was thought to be due mainly to the difference in cell populations of circulating myeloid cells between MDS patients and CML patients. Induction of NAP activity by rhG-CSF in patients with both these diseases was suppressed by the addition of inhibitors of RNA or protein synthesis. Neutrophil superoxide anion (O2- ) production induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was determined in the other 11 patients with MDS. This neutrophil function was decreased in seven of the 11 patients with MDS, normal in two patients, and increased in two patients. Preincubation with rhG-CSF caused a significant increase in fMLP-induced O2- production in nine of the 11 patients with MDS. rhG-CSF enhanced this neutrophil function in a time- and dose-dependent manner, and maximal stimulation was observed at 2,000 to 4,000 U/mL of rhG-CSF and at five to ten minutes' incubation. The present results show that rhG-CSF is able to repair at least in part the neutrophil anomalies in these patients, and our data, especially for patients with MDS, suggest the clinical usefulness of rhG-CSF for this preleukemic disorder.

The application of aqueous two-phase partition to the study of chick limb mesenchymal diversification

A technique which identifies cells differing in surface character, aqueous two-phase partition using thin-layer countercurrent distribution (TLCCD), has been used to study differentiation and pattern formation in the developing chick limb bud. The TLCCD profiles of cell populations, derived from various regions of morphologically undifferentiated mesenchyme from three different stages of limb development, have been compared. At no stage, or location, has the population been found to be homogeneous. Cells from progress zones and more proximal regions could all be resolved into several populations. The populations from progress zones at three different developmental stages were qualitatively similar but differed in the proportions of cells in each. The most striking differences in cell populations were those obtained from the most proximal region of the limb, closest to the flank, which represents the developmentally most advanced region.

Latency differences of lysosomal enzymes in cardiac and skeletal muscles of male and female mice

1. 1. The purpose of this study was to compare the latencies of lysosomal enzymes (β-glucuronidase, β- N-acetylglucosaminidase, arylsulphatase and acid ribonuclease) in heart and in red and white skeletal muscle of male and female mice ( Mus musculus). The unsedimentable, free activities together with releasable (Triton X-100, hypotonie shock and freeze-thawing treatments) and unreieasable, bound activities were assayed. 2. 2. The distribution of acid hydrolases to different fractions was strikingly heterogeneous. The most distinct differences occurred between the distributions of β-glucuronidase and β- N-acetylglucosaminidase. 3. 3. The differences between muscle types occurred in the activity levels of lysosomal enzymes, rather than in the fractional distributions. 4. 4. Sex-related differences were small and occurred mainly in the activity levels of heart muscle (higher in female mice). 5. 5. The results suggest that the heterogeneous distribution of lysosomaS enzymes originates in the compartimentai differences of lysosomal enzymes in muscle cells, rather than the differences in cell populations of different muscle types.

A quantitative method for the analysis of human periapical inflammation

The morphometric point-hit method for differential cell counting of periapical inflammation produces data comparable to thos of total differential cell counts. The technique reduces sample size by 79% and thus expedites quantitation. A modification of this technique in which only alternate microscopic fields are surveyed further reduces the cell count to 5.65% of the total population. The technique is not affected by differences in cell population and variations in cell distribution within a particular tissue section. Thus the method is practical, representative, sensitive, and suitable for cellular evaluation of human periapical inflammation.

Cyclic nucleotide metabolism during lymphocyte transformation: II. Comparative studies in the nude Balb/c mouse

Lymphocytes from the spleens of nude (athymic) Balb/c mice were incubated with LPS and examined for alterations in cyclic nucleotide metabolism. The cGMP concentration increased to 74% above control values within 20 min after the addition of LPS. cAMP concentration appeared to have two peaks, the first at 10 min to 80% above controls. The increase in cGMP can be accounted for by the inhibition of cGMP degradation by cAMP. These changes in cyclic nucleotide metabolism are similar to those observed in the “normal” Balb/c mouse. There are several differences between the cells from the two groups: (i) a delay in the cAMP and cGMP change and (ii) LPS enhanced cAMP inhibition of cGMP degradation in the nude mouse. These differences may be explained by differences in cell populations in the two mice, other than the obvious lack of T cells. Such differences were found on the basis of cell density distribution, where lymphocytes from normal Balb/c mice were found evenly dispersed between two dextran density gradient fractions, d = 1.057 and 1.066 g/ml; whereas, lymphocytes from nude mice were found almost entirely in the d = 1.066 g/ml fraction.