Proliferation and differentiation sequence of osteoblast histogenesis under physiological conditions in rat periodontal ligament

To define the mechanism of osteoblast histogenesis, nuclear morphometry was utilized as a marker for precursor cell differentiation. One hour after ³H-thymidine injection, groups of 7-week-old rats were killed at hourly intervals over one complete 24-hr photoperiod (LD 12:12). S-phase and mitosis were assessed in autoradiographs of 3-μm sections of molar periodontal ligament (PDL) adjacent to a physiological bone-forming surface. Labeled nuclei were divided into four categories according to morphometry of nuclear size: A (40–79 μm³), B (80–119 μm³), C (120–169 μm³), and D (≥ 170 μm³) cells. C and D cells synthesize DNA during the light and divide in the following dark phase; the rhythm for A cells is the opposite. B cells demonstrated no preference and were subsequently determined to be nonosteogenic. Compared to A cells the S-phase photoperiod of C and D cells (combined) is approximately a one-to-one reciprocal relationship, suggesting two proliferating progenitors in series. Based on arrest points in the histogenesis sequence, five compartments are defined: (1) A cells, less differentiated, self-perpetuating precursors; (2) A′ cells, committed osteoprogenitors; (3) C cells, G₁ stage preosteoblasts; (4) D cells, G₂ stage preosteoblasts; and (5) Ob cells, morphologically distinct osteoblasts. Minimal elapsed time for the A →A′ → C → D → Ob sequence is about 60 hr (five alternating dark/light cycles). A stress/strain-mediated increase in nuclear volume (A′ → C) is an important, rate-limiting step in osteoblast differentiation.     

A quantitative study of satellite cells and myonuclei in stretched avian slow tonic muscle

Satellite cell frequency was assessed in control and stretched anterior latissimus dorsi (ALD) muscles of adult quail. A weight equal to 10% of body mass was attached to one wing for time intervals ranging from 1–30 days. The contralateral ALD served as the intra-animal control. Satellite cell frequency, expressed as a percentage of total myofiber nuclei within the basal lamina, was determined in eight control and stretched ALD muscles, that had been weighted for 5, 7, or 10 days. Satellite cell frequency was determined in 584 control and 473 stretched fibers and was not different in the control or stretched ALD muscles (15.6 ± 2.3%, 16.7 ± 6.1%, respectively). The number of myofiber nuclei (myonuclei and satellite cell nuclei) was examined in whole fiber segments from control and stretched ALD muscles of 27 adult quail. Nuclear frequency was determined in 500 control and 1,200 stretched fiber segments. Fiber volume was calculated from fiber length and diameter measurements. Nuclear number normalized to 10,000 μm³ fiber volume was correlated to fiber cross-sectional area (P < 0.0001). Fibers with cross-sectional areas less than 500 μm² had a greater nuclear to fiber volume ratio compared to fibers with areas greater than 500 μm². The nuclear-to-cytoplasmic ratio was not constant in smaller fibers. Nuclear density decreased as fiber cross-sectional area increased up to 500 μm². Fibers with cross-sectional areas greater than 500 μm² demonstrated a constant nuclear-to-cytoplasmic ratio. The results demonstrate that absolute nuclear number increased to maintain a constant nuclear-to-cytoplasmic ratio in the stretched hypertrophied fibers. Daughter cells originating from activated satellite cells may have contributed to the myonuclear population to maintain a constant nuclear-to-cytoplasmic ratio in the hypertrophied fibers of the adult quail.     

Differential distribution of γ-aminobutyric acidA, receptor subunit (α1, α2, α3, α5 and β2+3) immunoreactivity in the medial prefrontal cortex of the rat

A detailed mapping of the γ-aminobutyric acid (GABA)_A receptor subunits (α₁, α₂, α₃ and β₂₊₃) in the infralimbic/ventral prelimbic region (IL/vPL) of the rat frontal cortex was carried out using subunit-specific antibodies. The α₁ and β₂₊₃ subunit antibodies immunostained all layers of the IL/vPL region. Layers II and III displayed immunostaining of cell bodies whereas I, V and VI showed predominantly neuropil staining. The size of the α₁-positive cell bodies corresponded to that of small interneurons (range, 20–55 μm²; mean ± SEM, 37 ± 5.5 μm²) as well as pyramidal cells or large interneurons (range, 87–135 μm²; mean ± SEM, 103.4 ± 9.7 μm²). However, β₂₊₃ antibody immunostained only small cell bodies. Immunoreactivity for α₂ was restricted to layers I and II, whereas α₃ and α₅ subunit expression was seen only in layer VI. The antibody to the α₂ subunit immunostained small cell bodies (range, 29–63 μm²; mean ± SEM, 32 ± 4.5 μm²) in layer II, resembling interneurons. Conversely, both α₃ and α₅ antibodies immunostained large cell bodies (range, 94–151 gmm²; mean ± SEM, 115.7 ± 13.4 μm²), consistent with pyramidal cell labelling in layer VI.     

Human uterine lower segment myometrial cell and nuclear volume at term: influence of maternal age

Little is known about the cytoarchitecture of human myometrial cells in pregnancy, and whether or not this may be influenced by maternal characteristics such as age, parity and body mass index (BMI). The aim of this study was primarily to evaluate human myometrial smooth muscle cell (SMC) and nuclear volume in the third trimester of human pregnancy, and secondarily to investigate if these parameters are altered in relation to the maternal characteristics outlined above. Myometrial biopsies were obtained from 30 women undergoing elective caesarean delivery at term. One‐micrometer sections were prepared for light microscopy and 100‐nm sections for electron microscopy. The nucleator technique was used to assess nuclear volume from the light microscopy images. Point‐counting methodology was used on transmission electron micrographs to assess the percentage of the cell volume occupied by the nucleus. Cell volume was calculated from these measurements. The euchromatin to heterochromatin (Eu/Het) ratio was determined to ascertain whether differences in nuclear volume were due to an increased range of genes being transcribed. The mean (± SEM) nuclear volume was 175 ± 10 μm³, the nucleus occupied 1.5 ± 0.1% of the SMC and the mean cell size was 14 047 ± 1352 μm³. The Eu/Het ratio was 7.54 ± 0.4. The mean volume of heterochromatin and euchromatin in the nucleus was 21.5 ± 1.7 and 149 ± 9 μm³, respectively. A multivariate regression analysis revealed that advanced maternal age was associated with an increase in the percentage of the cell occupied by nucleus (R² = 0.32, P = 0.004). There were no other significant effects of maternal age, BMI or parity on the measured parameters. These findings provide reliable volumes for human myometrial cells and their nuclei at term gestation, and show that nuclear volume fraction may be influenced by maternal age.     

Method for decreasing the statistical variance of stereological estimates

Three methods are described for decreasing the statistical variance of stereological estimates. Methods 1 uses profile boundaries and surface densities of nuclear membranes, measured in thin sections, to estimate the mean diameter, surface area, and numerical density of spherical and nonspherical nuclei. For the guinea pig pancreas (number(m) = 4), the standard deviations (s.d.) as a percent of the mean for the estimates of the diameters of the exocrine, duct, and endothelial cell nuclei were 1.5%, 3.3% and 1.4%. The estimate for the mean diameter of exocrine nuclei (6.4 ± 0.1 μm) was based on a spherical model, whereas the estimates for the diameters of the nonspherical (and nonconvex) nuclei of the duct (6.4 ± 0.2 μm) and endothelial (6.7 ± 0.1 μm) cells were calculated from the numerical density of the exocrine cells and the relative frequenceis of the three cell types (determined from serial reconstructions). In an average cubic centimeter, there were 6.17 × 10⁸ ± 0.32 × 10⁸ (s.d. 5.1% of mean) exocrine cells, 1.64 × 10⁸ ± 0.18 × 10⁸ (10.9%) duct cells, and 0.803 × 10⁸ ± 0.13 × 10⁸ (16.6%) endothelial cells. In contrast to method 1, conventional stereological approaches were found to have standard deviations two-to eightfold larger. Method 2 uses a mean nuclear surface area and a ratio of surface densities to estimate the surface area of a membrane compartment in an average cell. A s.d. equal to 6.5% of the mean was found for the surface are of the outer mitochondrial membrane in an average exocrine cell (672 ± 43.6 μm²), which represented an almost fourfold reduction in the s.d. compared with an earlier estimate (Bolender, 1974). Method 3 relates the surface area of a membrane compartment to a standard number of cells. Referenced to 10⁶ cells, for example, the surface area of the inner nuclear membrane of endothelial cells had a s.d. equal to 2.9% of the mean, whereas the surface density of the same membrane compartment—referenced to a cm³ to cells—had a s.d. at 19.1% of the mean. In this case, method 3 produced almost a sevenfold reduction in the standard deviation. Similar results were found for exocrine and duct cells. The results of the study indicate that the standard deviation of a stereological estimate can be reduced to a miniumum by using a mean nuclear profile boundary to generate an estimate for a nuclear numerical density, which, in turn, can be combined with a surface density to obtain average cell information.     

Distinct subpopulations of thymus-dependent lymphocytes: Tracing of the differentiation pathway of T cells by use of preparatively electrophoretically separated mouse lymphocytes

Thymus-dependent cells from thymus and peripheral lymphoid organs were preparatively separated by means of free flow electrophoresis into various subpopulations which were defined in terms of θ (theta) antigen content, negative surface charge, graft-versus-host (GvH) reactivity, hydrocortisone sensitivity, cell volume and morphological details. Most thymocytes in the cortex have a low negative surface charge, high θ antigen content, are hydrocortisone-sensitive and immuno-incompetent. On the basis of electronic cell sizing this group consists of a large population of 90 μm³ cells (T₁) and a small population of 175 μm³ cells (T₂), the latter being less hydrocortisone-sensitive than the former.

A minority of thymocytes resides in and around the medulla and has high negative surface charge, a medium θ antigen content, is hydrocortisone-resistant and reveals low GvH reactivity. These cells are medium sized (125 μm³), electrophoretically bimodal (T₃ had a medium and T₄ a high negative surface charge) and on the basis of morphological criteria are metabolically more active than the thymocytes of low negative surface charge.

In the peripheral lymphoid organs, all thymus-dependent cells show high negative surface charge and have the lowest observed θ antigen content and the highest observed GvH reactivity. These cells fall into two populations of which one is 125 μm³ with lower negative surface charge and the other is 90 μm³ with a somewhat higher negative surface charge. These 125 μm³ cells (T₄), which morphologically resemble the 125 μm³ thymocytes, are less GvH-reactive than the 90 μm³ (T₅) cells, which seem to be resting cells.

On the basis of these data, a possible sequence of steps in the maturation of T cells was constructed as follows: in the cortex of the thymus T₁ thymocytes are transformed into T₂ and these develop into T₃ and T₄ thymocytes which have higher negative surface charge, lower θ antigen content and are in an advanced stage of maturity. After further loss of θ antigen these cells, which are in the medulla, emigrate into the periphery and are finally transformed into highly immunocompetent T₅ cells possessing the highest observed negative surface charge.

     

Use of the optical disector in canine mammary simple and complex carcinomas

Grading of canine mammary carcinomas (CMC) is associated to subjective assessments made by the pathologists. Due to its unbiased nature, stereology can be used to objectively quantify morphological parameters associated with grading and malignancy. However, the use of stereology in CMC has not been fully disclosed. The nuclear numerical density [N_V (nuclei, tumor)] is a cellularity‐associated parameter that can be estimated by the optical disector. Herein, it was estimated in 44 CMC and its association with clinicopathologic factors – such as tumor size, histological subtype and grade, vascular/lymph node invasion, nuclear pleomorphism, and survival – was evaluated. Considering all the cases, the mean N_V (nuclei, tumor) was 1.6 × 10⁶ ± 0.5 × 10⁶ nuclei/mm³. Lower values were attained in complex carcinomas, comparing to simple carcinomas, in tumors smaller than 5 cm, with low mitotic activity and in those with high nuclear pleomorphism. No statistically significant association with grade or vascular/lymph node invasion was observed, but tumors with disease progression had lower nuclear densities. The N_V (nuclei, tumor) and the correlated parameters mirror to some extension those in human breast cancer, suggesting an interesting interspecies agreement. This first estimation of the nuclear numerical density in CMC highlights the feasibility of the optical disector and their utility for objective morphological assessments in CMC. The association between nuclear numerical density and disease progression warrants future studies.     

Fine-needle aspiration cytology diagnosis of colloid nodule versus follicular variant of papillary carcinoma of the thyroid

The cytologic differential diagnosis of colloid nodule (CN) and the follicular variant of papillary carcinoma (FVPC) is difficult with common morphologic features. To assess the utility of 18 cytologic morphometric parameters in the diagnosis of these thyroid lesions we evaluated 31 FNA samples that had histologic confirmation of the diagnoses. These 31 cases included 15 cases of CN, 8 cases of FVPC, and 8 cases of the usual variant of papillary carcinoma (UVPC) for reference values. For the morphometric analysis we used an Optimas 4.0 image analysis system. Comparing the CN group with the UVPC group revealed that eight of the parameters had statistically significant differences. The UVPC specimens were more cellular, less cohesive, had presence of papillary cellular groups more frequently, larger nuclei (UVPC: 109.33 ± 30.19 μm²; CN: 66.81 ± 15.02 μm2), higher nuclear to cytoplasmic (N/C) ratio, larger nucleoli, and present nuclear grooves and nuclear pseudoinclusions more frequently. The FVPC group differed from the CN group only in three parameters which included larger nuclei (98.49 ± 18.24 μm²), higher N/C ratio, and a more frequent presence of nuclear pseudoinclusions. When we compared these two variants of papillary carcinoma, we found that the UVPC specimens had less cellular cohesion, less preservation of the architectural polarity and a more frequent presence of papillary cellular groups than the FVPC. The FVPC can be differentiated from CN based on nuclear changes, which included a larger size, higher N/C ratio, and presence of pseudoinclusions. The absence of cellular cohesion and polarity combined with the presence of papillary groups are useful in separating the UVPC from the FVPC. A cutoff of 75 μm² should be used in separating benign from malignant nuclei. Diagn. Cytopathol. 1998;18:87–90. © 1998 Wiley-Liss, Inc.     

Osteoblast Interactions Within a Biomimetic Apatite Microenvironment

Numerous reports have shown that accelerated apatites can mediate osteoblastic differentiation in vitro and bone formation in vivo. However, how cells interact within the apatite microenvironment remains largely unclear, despite the vast literature available today. In response, this study evaluates the in vitro interactions of a well-characterized osteoblast cell line (MC3T3-E1) with the apatite microenvironment. Specifically, cell attachment, spreading, and viability were evaluated in the presence and absence of serum proteins. Proteins were found to be critical in the mediation of cell–apatite interactions, as adherence of MC3T3-E1 cells to apatite surfaces without protein coatings resulted in significant levels of cell death within 24 h in serum-free media. In the absence of protein–apatite interaction, cell viability could be “rescued” upon treatment of MC3T3-E1 cells with inhibitors to phosphate (PO₄ ³⁻) transport, suggesting that PO₄ ³⁻ uptake may play a role in viability. In contrast, rescue was not observed upon treatment with calcium (Ca²⁺) channel inhibitors. Interestingly, a rapid “pull-down” of extracellular Ca²⁺ and PO₄ ³⁻ ions onto the apatite surface could be measured upon the incubation of apatites with α-MEM, suggesting that cells may be subject to changing levels of Ca²⁺ and PO₄ ³⁻ within their microenvironment. Therefore, the biomimetic apatite surface may significantly alter the microenvironment of adherent osteoblasts and, as such, be capable of affecting both cell survival and differentiation.     

Correlation analysis of nuclear morphology,cytokeratin and Ki‐67 expression of urothelial carcinoma cells

We aimed to delineate the morphogenesis of aberrant nuclear features of urothelial carcinoma (UC) cells in association with cytokeratin (CK) expression patterns and cell proliferation activity. Correlation analysis of the nuclear area by morphometry and the expression patterns of CK5, CK20 and Ki‐67 by triple immunofluorescence analysis was applied to 1699 cells from five low‐grade and seven high‐grade cases of UC. The majority of UC cells showed aberrant cellular differentiation represented by abnormal CK expression patterns of CK5(+)/CK20(+) (40.5%) or CK5(?)/CK20(+) (56.0%). CK5(+)/CK20(?) cells, a phenotype of cancer stem/progenitor cells, represented a very small population (1.9%) and showed a low proliferation activity. Ki‐67(+) cells showed a significantly different CK expression pattern compared with that of Ki‐67(?) cells. The nuclear areas of CK5(?)/CK20(+) cells (71.3 ± 25.9 μm²) were significantly larger than those of CK5(+)/CK20(+) cells (66.6 ± 25.5 μm²). Negativity for CK5 was related to the grade of UC and an increased number of CK5(?)/CK20(+)/Ki‐67(+) cells was related to a higher malignant potential. We conclude the nuclear morphology is related to cell differentiation represented by CK expression and cell proliferative activity.     

Does stimulation of NaCl secretion in in vitro perfused rectal gland tubules of Squalus acanthias increase membrane capacitance?

 NaCl secretion in rectal gland tubules (RGT) of Squalus acanthias requires the activation of Cl^– channels in the luminal membrane. The RGT and its mechanism of activation are an early evolutionary paradigm of exocrine secretion. The respective Cl^– channels probably resemble the shark equivalent of the cystic fibrosis transmembrane conductance regulator (CFTR). Activation of these Cl^– channels occurs via cAMP. It has been hypothesized that the activation of CFTR occurs via exocytosis or inhibited endocytosis. To examine this question directly by electrical measurements we have performed whole-cell patch-clamp analyses of in vitro perfused RGT. NaCl secretion was stimulated by a solution (Stim) containing forskolin (10 μmol/l), dibutyryl-cAMP (0.5 mmol/l) and adenosine (0.5 mmol/l). This led to the expected strong depolarization and an increase in membrane conductance (G _m). The membrane capacitance (C _m) was measured by a newly devised two-frequency synchronous detector method. It was increased by Stim significantly from 5.00±0.22 to 5.17±0.21 pF (n=50). The increase in C _m correlated with the increase in G _m with a slope of 51 fF/nS. Next the effect of furosemide (500 μmol/l) was examined in previously stimulated RGT. Furosemide was supposed to inhibit coupled Na⁺2Cl^–K⁺ uptake and to reduce cell volume but not membrane trafficking of Cl^– channels. Furosemide reduced G _m slightly (due to the fall in cytosolic Cl^– concentration) and C _m to the same extent by which Stim had increased it. Both changes were statistically significant, and the slope of ΔC _m/ΔG _m was similar to that caused by Stim. Inhibitors of microtubules or actin (colchicine, phalloidin and cytochalasin D added at 10 μmol/l to the pipette solution and dialysed for >10 min) did not alter cell voltage, G _m or C _m, nor did these inhibitors abolish the stimulatory effect of cAMP. These data suggest that the small C _m changes observed with Stim reflect a minor cell volume increase and an ”unfolding” of the plasma membrane. The present data do not support the exocytosis/endocytosis hypothesis of cAMP-mediated activation of Cl^– channels in these cells. Received: 11 March 1998 / Received after revision 15 April 1998 / Accepted: 20 April 1998     

Quantitative histological analysis of the cerebellar nuclei in the cat. I. Numerical data on cells and on synapses

Summary The tissue volume, cell number, cell density, as well as the numbers and densities of various kinds of synaptic terminals were determined in the cerebellar nuclei of adult cats by means of stereological procedures both on the light and electron microscopic levels. The total number of the cerebellar nuclear cells was found to be 4.6×10⁴. On the basis of karyometric studies the medial and interpositus nuclei appear to contain two, the lateral nucleus probably three different neuron populations. The over-all numerical ratio between Purkinje and nuclear cells is 26:1.On the basis of simplified cytological and size criteria five different types of synaptic terminals were distinguished and counted separately. The total number of synaptic boutons was found to be 9.2×10⁸, 62% of which (5.7×10⁸) belong to Purkinje axons. The average number of synaptic boutons per nuclear cell is 2×10⁴ with systematic differences in the several nuclei (medial = 27500; interpositus = 18000; lateral = 13900). The number of boutons of Purkinje cell origin is 11600 per nuclear cell, on the average.The average number of synaptic boutons per Purkinje axon is 474, which are distributed in a space of about 13.5×10⁶ m³. In view of the density of the nuclear cells and the metric parameters of their dendrites, the number of nuclear cells with which synapses might be established is 35. This is a direct measure for the divergence; i.e. one Purkinje axon may reach potentially 35 nuclear cells. The number on any nuclear cell of boutons that originate from the same Purkinje axon would be mathematically 13.5 as an average, but may vary excessively between 1 and around 50 boutons. From these data the probable convergence of Purkinje axons upon nuclear cells can be deduced as being numerically somewhere around 860, however, this apparently excessive value is mitigated by the Golgi observation that a single Purkinje axon may contribute to the same nuclear cell as many as 50 somatic synapses. The dendritic synapses — forming the vast majority of all contacts — are probably more evenly distributed among the great majority of the converging Purkinje axons but with correspondingly fewer individual contacts.     

Immunohistochemical analyses of β-catenin and cyclin D1 expression in giant cell tumor of bone (GCTB): A possible role of Wnt pathway in GCTB tumorigenesis

Giant cell tumor of bone (GCTB) is a benign neoplasm but occasionally shows local recurrence, and histologically consists of osteoclast-like giant cells (GC) and stromal mononuclear cells (SC), which are capable of proliferation and osteoblastic differentiation. Activation of Wnt signaling can induce osteoblast differentiation and osteoclastgenesis during bone resorption process. This study analyzed the profiles of β-catenin and cyclin D1 expression in GCTB to elucidate an involvement of Wnt pathway in tumorigenesis. We performed immunohistochemistry for β-catenin, cyclin D1, and Ki-67 in 16 GCTB tumors, including 5 recurrent cases that were surgically resected. All 16 cases of GCTB displayed β-catenin, cyclin D1, and Ki-67 expression. Immunoreactivity for β-catenin was observed in nuclei of SC and GC. Cyclin D1 immunoreactivity was found mainly in nuclei of GC, while Ki-67 immunoreactivity was restricted to nuclei of SC. The nuclear β-catenin labeling index (LI) in both SC (60.6 vs. 41.8%, p=0.074) and GC (41.7 vs. 20.1%, p=0.095) was higher in recurrent tumors than in primary tumors in all the 4 cases. However, Ki-67 LI in SC (18.8 vs. 19.9%, p=0.851) and cyclin D1 LI in GC (55.4 vs. 70.1%, p=0.225) were not higher in recurrent tumors than in primary tumors. Our results suggested activation of Wnt/ β-catenin pathway in GCTB tumorigenesis. Since cyclin D1 in GC was never associated with the expression of the well-known proliferative marker Ki-67, cyclin D1 expression might play a role in GC formation instead of promoting cell proliferation during GCTB tumorigenesis. Importantly, it was suggested that the nuclear β-catenin staining level might be associated with tumor recurrence in GCTB.     

Hydrogen Peroxide Induces G2 Cell Cycle Arrest and Inhibits Cell Proliferation in Osteoblasts

Reactive oxygen species (ROSs) are involved in osteoporosis by inhibiting osteoblastic differentiation and stimulating osteoclastgenesis. Little is known about the role and how ROS controls proliferation of osteoblasts. Mammalian target of rapamycin, mTOR, is a central regulator of cell growth and proliferation. Here, we report for the first time that 5–200 μM hydrogen peroxide (H₂O₂) dose‐ and time‐dependently suppressed cell proliferation without affecting cell viability in mouse osteoblast cell line, MC3T3‐E1, and in human osteoblast‐like cell line, MG63. Further study revealed that protein level of cyclin B1 decreased markedly and the percentage of the cells in G₂/M phase increased about 2‐4 fold by 200 μM H₂O₂ treatment for 24–72 hr. A total of 0.5–5 mM of H₂O₂ but not lower concentrations (5–200 μM) of H₂O₂ inhibited mTOR signaling, as manifested by dephosphorylation of S6K (T389), 4E‐BP1 (T37/46), and S6(S235/236) in MC3T3‐E1 and MG63 cells. Rapamycin, which could inhibit mTOR signaling and cell proliferation, however, did not reduce the protein level of cyclin B1. In a summary, H₂O₂ prevents cell proliferation of osteoblasts by down‐regulating cyclin B1 and inducing G₂ cell cycle arrest. Inhibition of mTOR signaling by H₂O₂ may not be involved in this process. Anat Rec, 292:1107–1113, 2009. © 2009 Wiley‐Liss, Inc.     

The histogenesis of the isthmic nuclei in chick embryos (Gallus domesticus)

Summary The neuromeric mes-rhombencephalic boundary runs between the oculomotor and trochlear nuclei. During morphogenesis the m ₂-segment is reduced to a cellfree mantle demarcating a morphological mes-rhombencephalic border. The floor plate and glycogen-containing raphe extend rostralwards to terminate at the level of the m ₂-segment ventromedially.The isthmic migration commences within the dorsal and dorsolateral rhombencephalic cell columns caudal to the emergence of the trochlear nerve. The neuroblasts migrate radially out from the synthetic zone of the neural epithelium into the mantle constituting the isthmic migration. The latter migrate longitudinally en masse rostralwards into the mantle below the optic lobe. The m ₂-segment can, however, be identified as a morphological border between the mesencephalic and isthmic (rhombencephalic) mantles throughout the early embryogenesis.The isthmic migration subdivides into a tectal and a tegmental nuclear group. Both groups contribute to the formation of the isthmic nuclei. The caudal portion of the mesencephalic tectal mantle contributes to a mesencephalic isthmic nucleus: Nucleus isthmi principalis mesencephali (magnocellularis).     

K+ channel antisense oligodeoxynucleotides inhibit cytokine-induced expansion of human hemopoietic progenitors

Primitive human hemopoietic progenitor cells identified by surface membrane markers CD33^–CD34⁺ are capable of expansion into lineage-restricted precursors following in vitro stimulation by hemopoietic regulators such as stem cell factor (SCF) and interleukin-3 (IL-3). In search of ionic currents involved in cytokine-induced progenitor cell growth and differentiation, human umbilical cord blood CD33^–CD34⁺ cells were subjected to perforated patch-clamp recordings following overnight incubation with SCF and/or IL-3. An inward rectifying potassium channel (K_ir) was found in 33% of control unstimulated cells, in 34% of cells incubated with IL-3, in 31 % of cells incubated with SCF and in 75% of cells incubated with IL-3 plus SCE K_ir activity increased with elevation of extracellular potassium and was blocked by extracellular Cs⁺ or Ba²⁺. Antisense oligodeoxynucleotides directed against K_ir blocked both mRNA and functional expression of K_ir channels. K_ir antisense also inhibited the in vitro expansion of cytokine-stimulated CD33^–CD34⁺ cells into erythroid (BFU-E) and myeloid (GM-CFU) progenitors in 7-day suspension cultures. Extracellular Cs⁺ or Ba²⁺ induced a similar degree of inhibition (40–60%) of progenitor cell generation. These findings strongly suggest an essential role for K_ir in the process of cytokine-induced primitive progenitor cell growth and differentiation.     

Decidual cells in the human ovary at term: II. Morphometric analysis of cytoplasmic processes and organelles

Morphometric analysis of human ovarian decidual cells was performed with a Videoplan computer, and mean values were established for the area and perimeter of cellular processes and organelles. Two-hundred forty electron micrographs representing 160 cells were analyzed. The mean decidual cell area was 218.7 μm² of which 34.5 μm² was occupied by the nucleus (15.8% of the cytoplasmic area); the nucleus contained 1.74 μm² of nucleolar material (0.8%). The endoplasmic reticulum occupied 13.63 μm² (6.2%). Mitochondria occupied 7.3 μm² (3.3%) and the Golgi network 5.49 μm² (2.5%). Decidual secretory bodies occupied 0.91 μm² (0.42%) and cytoplasmic processes 1.89 μm² (0.94%). The remainder of the cytoplasm, containing inclusions and cytoskeleton, represented 71% of the cell area. Perimeter measurements indicated an average decidual cell was surrounded by 87.8 μm of plasma membrane. The mean nuclear membrane measured 28.3 μm (representing 32.3% of the plasma membrane, pm, or 4.1% of total cellular membranes, cm). Outer mitochondrial membranes measured 156.6 μm (178% pm, 23.5% cm); endoplasmic reticulum membranes measured 350.3 μm (400% pm, 52.6% cm); Golgi membrane measured 30.77 μm (35% pm; 4.5% cm) and membrane surrounding secretory bodies measured 9.8 μm (11.2% pm; 1.4% cm). A mean of 280 secretory bodies per ovarian decidual cell was calculated. The plasma membranes of evaginated cytoplasmic processes represented 22.3% of the total pm (19.6 μm or 2.9% cm). A mean of seven such processes was observed per 87.8 μm of plasma membrane (160/cell). These morphometric data provide a baseline for comparisons of human ovarian decidual cells with uterine decidua, in vivo and in vitro, as well as with decidual cells of other species.     

Treatment with Tumor Necrosis Factor-α and Granulocyte–Macrophage Colony-Stimulating Factor Increases Epidermal Langerhans' Cell Numbers in Cancer Patients

Dendritic cells (DCs) initiate primary and stimulate secondary T-cell responses. We conducted a phase I trial of tumor necrosis factor (TNF-α) and granulocyte–macrophage colony-stimulating factor (GM-CSF) in patients with cancer to increase DCs in peripheral blood or skin based on in vitro data that showed that CD34⁺ hematopoietic precursors require these cytokines to mature into functional antigen-presenting DCs. Eleven patients were treated for 7 days with GM-CSF, 125 μg/m² twice daily as subcutaneous injections, and TNF-α as a continuous infusion at dose levels of 25, 50, or 100 μg/m²/day. The maximum tolerated dose of TNF-α was 50 μg/m²/day with this dose of GM-CSF; dose-limiting toxicities occurred in both patients treated with 100 μg/m²/day. One became thrombocytopenic and the other had transient confusion. Epidermal Langerhans' cells were quantitated by S100 staining of skin biopsies and DC precursors in peripheral blood by colony-forming unit dendritic (CFU-dendritic) assays. S100-positive cells in the epidermis doubled after treatment (2.55 S100⁺ cells/high-power field before treatment to 6.05 after treatment, p = 0.029). CFU-dendritic in peripheral blood increased after treatment in 3 colorectal cancer patients but not in 3 patients with melanoma. CD11c⁺ or CD123⁺, HLA-DR^bright, lineage-negative dendritic cell precursors were not increased in peripheral blood mononuclear cells. This trial demonstrates that treatment with TNF-α and GM-CSF can increase the number of DCs in the skin and the number of dendritic cell precursors in the blood of some patients with cancer. This approach may increase the efficacy of vaccination to tumor antigens in cancer patients.     

Corticosteroids reverse cytokine-induced block of survival and differentiation of oligodendrocyte progenitor cells from rats

Background

Periventricular leukomalacia (PVL) is a frequent complication of preterm delivery. Proinflammatory cytokines, such as interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) released from astrocytes and microglia activated by infection or ischemia have previously been shown to impair survival and maturation of oligodendrocyte progenitors and could thus be considered as potential factors contributing to the generation of this disease. The first goal of the present study was to investigate whether exposure of oligodendrocyte precursors to these cytokines arrests the maturation of ion currents in parallel to its effects on myelin proteins and morphological maturation. Secondly, in the search for agents, that can protect differentiating oligodendrocyte precursor cells from cytokine-induced damage we investigated effects of coapplications of corticosteroids with proinflammatory cytokines on the subsequent survival and differentiation of oligodendrocyte progenitor cells.

Methods

To exclude influences from factors released from other cell types purified cultures of oligodendrocyte precursors were exposed to cytokines and/or steroids and allowed to differentiate for further 6 days in culture. Changes in membrane surface were investigated with capacitance recordings and Scanning Ion Conductance Microscopy. Na⁺- and K⁺- currents were investigated using whole cell patch clamp recordings. The expression of myelin specific proteins was investigated using western blots and the precursor cells were identified using immunostaining with A2B5 antibodies.

Results

Surviving IFN-γ and TNF-α treated cells continued to maintain voltage-activated Na⁺- and K⁺ currents characteristic for the immature cells after 6 days in differentiation medium. Corticosterone, dihydrocorticosterone and, most prominently dexamethasone, counteracted the deleterious effects of IFN-γ and TNF-α on cell survival, A2B5-immunostaining and expression of myelin basic protein. The most potent corticosteroid tested, dexamethasone, was shown to counteract cytokine effects on membrane surface extension and capacitance. Furthermore, coapplication of dexamethasone blocked the cytokine-induced downregulation of the inwardly rectifying potassium current in 80% of the precursor cells and restored the cytokine-blocked down-regulation of the voltage activated Na⁺- and K⁺ currents during subsequent differentiation.

Conclusion

Our results show that treatment of oligodendrocyte precursors with the inflammatory cytokines TNF-α and IFN-γ block the differentiation of oligodendrocyte precursors at the level of the differentiation of the voltage-gated ion currents. Co-treatment with corticosteroids at the time of cytokine application restores to a considerable extent survival and differentiation of oligodendrocytes at the level of morphological, myelin protein as well as ion current maturation suggesting the option for a functional restoration of cytokine-damaged immature oligodendrocytes.