Large Cell Aggregates Research Articles

Dielectric spectroscopy of blood

An experimental investigation of complex dielectric spectra of human blood in the frequency range 30 Hz–30 MHz has been carried out for various blood cell concentrations, temperature and environment. Electrode impedances and other distorting effects have been excluded. The average values of the dielectric constant (DC) ε and conductivity γ of normal heparinized blood for the lowest ( ε 0, γ 0) and the highest frequencies ( ε ∞, γ ∞) are: ε 0=7500; γ 0=4.9 mS/cm; ε ∞=160; γ ∞=9 mS/cm. The characteristic relaxation time τ=0.265 μ s with a Cole–Cole distribution around it; only beta-dispersion is observed in normal blood. The DC of resting blood is considerably larger than that of stirred blood; this is associated with the formation of small aggregates of blood cells. The temperature dependence of the static DC shows a maximum at the temperature 37 °C. According to Fricke–Pauly–Schwan theory the membrane capacity C m of stirred blood at normal concentration is 0.8 μF/cm 2 and depends strongly on concentration of cells and stirring. Damaged and aged cells manifest, in addition to beta-dispersion, a low-frequency alpha-dispersion, which can be explained as a result of the formation of large aggregates of cells.

Conserved serine/threonine kinase encoded by CBK1 regulates expression of several hypha-associated transcripts and genes encoding cell wall proteins in Candida albicans.

The opportunistic fungal pathogen, Candida albicans, is reported to have several potential virulence factors. A potentially significant factor is the ability to undergo morphological transition from yeast to hypha. This alteration of form is accompanied by many changes within the cell, including alterations in gene expression and cell wall composition. We have isolated a gene that encodes a highly conserved serine/threonine kinase that appears to be involved in the regulation of proteins associated with the cell wall. We have assigned the designation CBK1 (cell wall biosynthesis kinase 1) to this gene. Mutants lacking CBK1 form large aggregates of round cells under all growth conditions and lack the ability to undergo morphological differentiation. Additionally, these mutants show an altered pattern of expression of several transcripts encoding proteins associated with the cell wall. The results suggest that the kinase encoded by CBK1 plays a general role in the maintenance and alteration of the cell wall of C. albicans in all morphologies.

Platelet-platelet and platelet-leukocyte interactions induced by outer membrane vesicles from N. meningitidis

A large part of native meningococcal lipopolysaccharide (LPS), i.e., LPS integrated in the outer cell membrane, is released in the form of 'blebs' from surplus outer membrane material. In the present study we investigated the effects of purified outer membrane vesicles (OMVs) on blood platelet-platelet and platelet-leukocyte interactions. Citrated whole blood was stimulated in vitro with equal amounts (on a weight basis) of OMV-integrated LPS, purified LPS (P-LPS) from the same meningococcal strain and purified E. coli -LPS. The samples were analyzed by flow cytometry. Upon OMV stimulation platelet aggregation increased 2.1-fold, platelet degranulation 1.8-fold, (measured as CD62P expression), platelet binding to monocytes 2.6-fold, whereas platelet binding to granulocytes increased 2.8-fold. Also, the fraction of large heteroconjugates, i.e., large CD45-positive cell aggregates increased 15.7-fold compared to control. P-LPS and E. coli -LPS also significantly increased platelet aggregation and heteroconjugate formation but did not influence platelet degranulation and binding of platelets to leukocytes in whole blood. When using platelet-rich plasma (PRP), OMVs increased platelet aggregation 2.1-fold and CD62P expression 1.9-fold. P-LPS and E. coli -LPS also significantly increased platelet aggregation in PRP but did not influence platelet degranulation. None of the LPS preparations induced platelet microvesiculation, either in whole blood or in PRP. Conclusion: Meningococcal-derived OMVs as well as purified meningococcal LPS, contribute to increased platelet-platelet and platelet-leukocyte aggregation and may thus be of great importance in the development of microthrombosis and organ dysfunction related to fulminant meningococcal septicemia.

Effect of Mixing Time on Taxoid Production Using Suspension Cultures of Taxus chinensis in a Centrifugal Impeller Bioreactor

The effects of fluid mixing on the cell growth and secondary metabolite production of plant cells were investigated in a low-shear centrifugal impeller bioreactor (CIB) system. Suspension cultures of Taxus chinensis cells producing taxuyunnanine C (Tc), a physiologically active secondary metabolite, were used as a model system for this investigation. The mixing time (t(m)) and volumetric oxygen transfer coefficient (k(L)a) in the bioreactor were characterized at various cell densities and operating conditions. A constant t(m) of 5 s or 10 s was maintained during cultivation by adjusting the impeller agitation speed with no detrimental effect on the cultured cells. A higher cell density, Tc content and total Tc production were obtained under the shorter mixing time of 5 s. The favorable effect of more rapid mixing on Tc production was also confirmed when the Tc accumulation was significantly increased through culture elicitation using 100 microM methyl jasmonate (MJA). The lower Tc production at the longer t(m) of 10 s was mainly attributed to oxygen transfer limitation in the dead zones and larger cell aggregates resulting from poor mixing.

Comparison of three in vitro human 'angiogenesis' assays with capillaries formed in vivo.

Angiogenesis assays are an important tool for studying both the mechanisms of angiogenesis and the potential development of therapeutic strategies to modulate neovascularisation. In vivo angiogenesis assays are considered to be the most informative of these but are often expensive, time-consuming and require specialist training to perform. In vitro assays tend to be more rapid, less expensive and easier to interpret. In vitro angiogenesis assays operate on the principle that endothelial cells form tubule-like structures when cultured on a supportive matrix. Assays involving a matrix derived from murine tumours, Matrigel (or a growth factor reduced form of this), are now the most common in vitro tubule formation assays. However, another tubule formation assay has recently been developed in which endothelial cells are co-cultured with fibroblasts. Here, we have used quantitative image analysis to compare the morphological features of tubules formed in the Matrigel assay and this new 'Co-culture' assay, with those of capillaries formed in a microvascular bed in vivo. Tubules formed in standard and growth factor reduced Matrigel assays were short and relatively homogeneous, whereas those formed in the Co-culture assay were significantly more heterogeneous, consisting of both short and long interconnecting tubules that more closely resembled capillaries than Matrigel tubules. Moreover, cells on Matrigel, and to a lesser extent growth factor reduced (GFR) Matrigel, often clumped into large cell aggregates, a feature rarely seen in the Co-culture assay. In addition, we demonstrate that Matrigel stimulates tubule formation by various non-endothelial cell types, suggesting that tubule formation by endothelial cells may not represent true differentiation of this cell type. In summary, the morphology of tubules in the Co-culture assay appears more representative of capillary formation in vivo, than the endothelial cell changes that occur in either form of Matrigel assay.

Cbk1p, a protein similar to the human myotonic dystrophy kinase, is essential for normal morphogenesis in Saccharomyces cerevisiae.

We have studied the CBK1 gene of Saccharomyces cerevisiae, which encodes a conserved protein kinase similar to the human myotonic dystrophy kinase. We have shown that the subcellular localization of the protein, Cbk1p, varies in a cell cycle-dependent manner. Three phenotypes are associated with the inactivation of the CBK1 gene: large aggregates of cells, round rather than ellipsoidal cells and a change from a bipolar to a random budding pattern. Two-hybrid and extragenic suppressor studies have linked Cbk1p with the transcription factor Ace2p, which is responsible for the transcription of chitinase. Cbk1p is necessary for the activation of Ace2p and we have shown that the aggregation phenotype is due to a lack of chitinase expression. The random budding pattern and the round cell phenotype of the CBK1 deletion strain show that in addition to its role in regulating chitinase expression via Ace2p, Cbk1p is essential for a wild-type morphological development of the cell.

Nonlymphoid intraepidermal mononuclear cell collections (pseudo-Pautrier abscesses): a morphologic and immunophenotypical characterization.

We evaluated the incidence, morphology, and immunophenotype of intraepidermal collections of mononuclear cells (ICMC) in a large number of inflammatory dermatosis and cutaneous lymphomas. ICMC appeared as small to large aggregates of cells, showing a morphology variable from monocytes to obvious dendritic cells, admixed with rare lymphocytes. ICMC were recognized in the epidermis or within hair follicle epithelium, and were either loosely or compactly arranged. ICMC were identified in 124 of 1,248 skin biopsies (9.9%) of inflammatory or lymphoid infiltrates, and were particularly frequent in spongiotic (43.4%) and in lichenoid dermatitis (10%), whereas they were rarely found in nonspecific superficial dermatitis (3.8%) and in psoriasis (4.7%). ICMC were also frequent in cutaneous T-cell lymphoma (13.3%), where they mimicked Pautrier abscesses. The ICMC forming cells showed a unique phenotype: the majority of them expressed CD1a and S-100, and lacked CD14, similar to mature Langerhans cells, but they were also strongly labeled by anti-CD11b, anti-CD36, and anti-CD68. Moreover, a subpopulation of them expressed CD83, an antigen that is usually absent on Langerhans cells. The occurrence of ICMC is a rather frequent, although hitherto poorly studied, phenomenon, occurring in several dermatosis, but particularly frequent in spongiosis-associated skin reactions. The cells within ICMC are represented by dendritic cells and dendritic cell precursors, whose phenotype indicates their derivation from circulating monocytes and differentiation into mature Langerhans cells.

Salivary Duct Carcinoma in Five Cats

Carcinomas1999 Harcourt Publishers Ltdof salivary gland ducts are described in five cats. The typical histological pattern was the formation of large cell aggregates resembling dilated ducts, often with central necrosis and a looping pattern. All tumours were labelled with antibody to cytokeratins (CKs) 5, 6, 8, 14, 17 and 19. Labelling of tumour cells with CK14 suggested basal cell differentiation. All tumours stained with Jack bean (Copyright Canavalia ensiformis) agglutinin (Con A); this is a feature of normal salivary gland ducts but is seen in other salivary gland tumours. Staining of tumour cells at the luminal surface of ductal structures with wheat germ (Triticum vulgaris) agglutinin (WGA) in the cat tumours was similar to that seen in ducts of normal cat salivary glands but occurs in other cat tumours. Other immunohistochemical staining results were unremarkable. 1999 W.B. Saunders and Company Ltd.

Cardiac organogenesis in vitro: reestablishment of three-dimensional tissue architecture by dissociated neonatal rat ventricular cells.

The mammalian heart does not regenerate in vivo. The heart is, therefore, an excellent candidate for tissue engineering approaches and for the use of biosynthetic devices in the replacement or augmentation of defective tissue. Unfortunately, little is known about the capacity of isolated heart cells to re-establish tissue architectures in vitro. In this study, we examined the possibility that cardiac cells possess a latent organizational potential that is unrealized within the mechanically active tissue but that can be accessed in quiescent environments in culture. In the series of experiments presented here, total cell populations were isolated from neonatal rat ventricles and recombined in rotating bioreactors containing a serum-free medium and surfaces for cell attachment. The extent to which tissue-like structure and contractile function were established was assessed using a combination of morphological, physiological, and biochemical techniques. We found that mixed populations of ventricular cells formed extensive three-dimensional aggregates that were spontaneously and rhythmically contractile and that large aggregates of structurally-organized cells contracted in unison. The cells were differentially distributed in these aggregates and formed architectures that were indistinguishable from those of intact tissue. These architectures arose in the absence of three-dimensional cues from the matrix, and the formation of organotypic structures was apparently driven by the cells themselves. Our observations suggest that cardiac cells possess an innate capacity to re-establish complex, three-dimensional, cardiac organization in vitro. Understanding the basis of this capacity, and harnessing the organizational potential of heart cells, will be critical in the development of tissue homologues for use in basic research and in the engineering of biosynthetic implants for the treatment of cardiac disease.

Regulation and possible function of axl expression in immature human mast cells.

The receptor tyrosine kinase Axl which expresses extracellular domains reminiscent of cell adhesion molecules, is involved in homotypic binding as well as in intracellular signaling of myeloid progenitor cells. In order to investigate factors which might influence differentiation pathways through changes of the adhesive properties of cells, we analyzed the expression of axl in immature basophil and mast cell lines and in cultured basophil and mast cell precursors. Axl expression was induced by interferon-alpha in the human leukemic mast cell line HMC-1 and in cultured mast cells derived from CD34+ peripheral blood cells. Axl induction was dose dependent, appeared within 1 h, and was independent of de novo protein synthesis. IFNalpha-treated HMC-1 cells expressing axl formed large cell aggregates within 40 h while untreated cells did not. HMC-1 cells also expressed gas6, the putative ligand of axl, which has been shown to induce axl-mediated homotypic binding. Gas6 expression was independent of interferon treatment in HMC-1 cells. The present results suggest that axl-mediated changes of cellular adhesive properties in mast cells may be important in mast cell differentiation as well as in mast cell-associated inflammation.

Insulin-like growth factor-I-mediated survival from anoikis: role of cell aggregation and focal adhesion kinase.

Anoikis is a form of cell death that occurs when cells are denied attachment to the extra-cellular matrix. Using p6 cells, that are 3T3 cells overexpressing the type 1 insulin-like growth factor receptor (IGF-IR), we show that these cells undergo apoptosis when seeded on polyHEMA plates in serum-free medium (SFM). IGF-I protects p6 cells from anoikis, without inducing mitogenesis or DNA synthesis. In the surviving p6 cells in suspension cultures, the focal adhesion kinase (FAK) is tyrosyl phosphorylated by IGF-I, although this phosphorylation occurs only after several hours. The importance of FAK in protection from anoikis is confirmed by v-src-transformed R-cells, in which FAK is constitutively phosphorylated, that survive even in SFM. Surviving cells, whether p6 or v-src transformed, tend to form large cell aggregates, whose appearance precedes the phosphorylation of FAK. These and other findings suggest that FAK phosphorylation in the case of IGF-I is a mediated effect rather than a direct one. When p6 cells are plated on polyHEMA dishes, IGF-I induces cell aggregation and this aggregation correlates with survival and the eventual phosphorylation of FAK.

Insulin‐like growth factor‐I‐mediated survival from Anoikis: Role of cell aggregation and focal adhesion kinase

Anoikis is a form of cell death that occurs when cells are denied attachment to the extra-cellular matrix. Using p6 cells, that are 3T3 cells overexpressing the type 1 insulin-like growth factor receptor (IGF-IR), we show that these cells undergo apoptosis when seeded on polyHEMA plates in serum-free medium (SFM). IGF-I protects p6 cells from anoikis, without inducing mitogenesis or DNA synthesis. In the surviving p6 cells in suspension cultures, the focal adhesion kinase (FAK) is tyrosyl phosphorylated by IGF-I, although this phosphorylation occurs only after several hours. The importance of FAK in protection from anoikis is confirmed by v-src-transformed R-cells, in which FAK is constitutively phosphorylated, that survive even in SFM. Surviving cells, whether p6 or v-src transformed, tend to form large cell aggregates, whose appearance precedes the phosphorylation of FAK. These and other findings suggest that FAK phosphorylation in the case of IGF-I is a mediated effect rather than a direct one. When p6 cells are plated on polyHEMA dishes, IGF-I induces cell aggregation and this aggregation correlates with survival and the eventual phosphorylation of FAK. J. Cell. Physiol. 176:648–657, 1998. © 1998 Wiley-Liss, Inc.

Behavior of CHO cells on phosphated cellulose membranes.

Phosphate groups (negatively charged chemical groups) were grafted onto the surface of cellulose membranes by a reaction between hydroxyl groups of cellulose and phosphorus pentoxide to observe the effect of phosphate groups on cellular behavior. X-ray photoelectron spectroscopy (XPS) was used to determine phosphorylation. Captive bubble contact angle measurement was used to determine surface wettability. XPS was also used to analyze serum protein adsorption. Chinese hamster ovary (CHO) cells were maintained in Ham's F-12 nutrient mixture with and without fetal calf serum. Total cell area and shape factor were analyzed using image-analyzing software. Serum proteins showed higher adsorption on phosphated cellulose. Cell spreading on phosphated membranes was greater than on the cellulose membrane that served as control. The cell growth rate was faster compared to the control. Large cell aggregates were not found on the phosphated membranes, in contrast to the control membrane. The cells on the control were aggregated regardless of the existence of divalent cations in the medium.

A quorum-sensing system in the free-living photosynthetic bacterium Rhodobacter sphaeroides.

Rhodobacter sphaeroides is a free-living, photoheterotrophic bacterium known for its genomic and metabolic complexity. We have discovered that this purple photosynthetic organism possesses a quorum-sensing system. Quorum sensing occurs in a number of eukaryotic host-associated gram-negative bacteria. In these bacteria there are two genes required for quorum sensing, the luxR and luxI homologs, and there is an acylhomoserine lactone signal molecule synthesized by the product of the luxI homolog. In R. sphaeroides, synthesis of a novel homoserine lactone signal, 7,8-cis-N-(tetradecenoyl)homoserine lactone, is directed by a luxI homolog termed cerI. Two open reading frames immediately upstream of cerI are proposed to be components of the quorum-sensing system. The first of these is a luxR homolog termed cerR, and the second is a small open reading frame of 159 bp. Inactivation of cerI in R. sphaeroides results in mucoid colony formation on agar and formation of large aggregates of cells in liquid cultures. Clumping of CerI mutants in liquid culture is reversible upon addition of the acylhomoserine lactone signal and represents a phenotype unlike those controlled by quorum sensing in other bacteria.

Acinar differentiation by non-malignant immortalized human prostatic epithelial cells and its loss by malignant cells.

Invasive prostatic carcinomas and prostatic intraepithelial neoplasia (PIN) are characterized by a loss of normal cell organization, cell polarity, and cell:cell and cell:basement membrane adhesion. The objective of this study was to establish in vitro three-dimensional (3-D) cell models which can be used to investigate mechanisms involved in acinar morphogenesis and differentiation in normal prostatic epithelium and their abnormalities in cancer cells. The process of acinar morphogenesis, including structural and functional differentiation, was investigated by culture on basement membrane gels (Matrigel). The human papillomavirus 18 immortalized, non-tumorigenic cell line RWPE-1, the v-Ki-ras transformed, tumorigenic RWPE-2 cell line derived from RWPE-1 cells (see previous paper pp. 1221-1229) and the human prostatic carcinoma cell line DU-145 were used. When cultured on Matrigel, RWPE-2 cells remain as single cells or form small aggregates and DU-145 cells form large amorphous cell aggregates without any organization or lumen. In contrast, RWPE-1 cells form acini of polarized epithelium with a distinct lumen, show a distinct laminin basement membrane, and express alpha6beta1 integrins at their basal end. Exposure to conditioned medium from NIH 3T3 cultures accelerates glandular morphogenesis. Parallel cultures maintained as monolayers on plastic remain as monolayers. In the presence of the synthetic androgen mibolerone, acinar cells express prostate specific antigen (PSA) as determined by immunostaining. We conclude that normal prostate cells can undergo acinar morphogenesis while tumorigenic cells have lost this ability. The 3-D cultures provide physiologically relevant in vitro models for elucidating regulation of growth, morphogenesis and differentiation in the normal human prostate, for defining heterotypic interactions in benign prostatic hyperplasia and for establishing the basis for the loss of normal cell organization in early neoplastic lesions such as PIN as well as during tumor progression in prostate cancer.

Islet morphogenesis and stem cell markers in rat pancreas.

During embryonic development, and possibly also later in life, pancreatic islets of Langerhans originate from differentiating epithelial stem cells. These stem cells are situated in the pancreatic ducts but are otherwise poorly characterized. We found by immunohistochemical staining that protodifferentiated pancreatic epithelial cells from rat embryos of Day 13-Day 15 express the cytoskeletal protein keratin 20, similar to mature duct epithelium. During the period of islet morphogenesis, which occurs between Day 17 and birth, large aggregates of K20-positive duct cells were formed, which gradually differentiated into endocrine cells. This islet morphogenic mechanism has not been described thus far and we did not observe it in postnatal rats. During fetal islet formation, transient expression of vimentin was noted in the duct cells but not in endocrine cells. This intermediate filament protein is not observed in duct epithelial cells after birth. The proto-oncogene product bcl-2, a putative epithelial stem cell marker, was detected in duct cells from fetal and postnatal pancreas. We conclude that K20, vimentin, and bcl-2 are markets for the pancreatic (islet) stem cells.

Ligation of TAPA-1 (CD81) or major histocompatibility complex class II in co-cultures of human B and T lymphocytes enhances interleukin-4 synthesis by antigen-specific CD4+ T cells.

We have previously shown that CD4+ T cells from allergic individuals are predisposed to producing interleukin (IL)-4 in response to allergens. IL-4 production could be modulated by antigen concentration as well as by the type of antigen-presenting cells (APC), with B lymphocytes inducing greater quantities of IL-4 than monocytes. Using this system we examined IL-4 synthesis after culture of CD4+ T cells with B cells, monocytes, or both, as APC in the presence of allergen and a monoclonal antibody against CD81 (TAPA-1), a member of the TM4 superfamily of proteins that regulates activation, proliferation and trafficking of B cells. Addition of anti-CD81 mAb during culture enhanced IL-4 synthesis by 2- to 70-fold over that using an isotype-matched control mAb. Furthermore, anti-CD81 mAb enhanced IL-4 synthesis in CD4+ T cells only when CD4+ T cells were cultured with B cells but not monocytes as APC, indicating that anti-CD81 mAb affected IL-4 synthesis in T cells via interactions with B cells. However, pretreatment of either population separately with anti-CD81 mAb prior to culture had no effect on subsequent IL-4 synthesis, suggesting a requirement for temporal or cooperative interactions between T and B lymphocytes. In addition, anti-CD81 mAb enhanced IL-4 production but reduced CD4+ T cell antigen-specific proliferation, demonstrating that IL-4 production and proliferation by CD4+ T cells were inversely related. Finally, mAb to major histocompatibility complex class II but not to anti-CD19 also enhanced IL-4 synthesis when B lymphocytes were used as APC. In all instances, enhancement of CD4+ IL-4 synthesis correlated with the presence of large cell aggregates in T-B lymphocyte cocultures. These results indicate that the capacity of B cells to induce IL-4 can be significantly enhanced by ligation of particular molecules on their surface and should aid in the design of treatments for diseases in which modulation of the cytokine profile would be beneficial.

Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis.

The Staphylococcus epidermidis genes icaABC are involved in the synthesis of the polysaccharide intercellular adhesin (PIA), which is located mainly on the cell surface, as shown by immunofluorescence studies with PIA-specific antiserum. PIA was shown to be a linear beta-1,6-linked glucosaminoglycan composed of at least 130 2-deoxy-2-amino-D-glucopyranosyl residues of which 80-85% are N-acetylated, the rest being non-N-acetylated and positively charged. A transposon insertion in the icaABC gene cluster (ica, intercellular adhesion) led to the loss of several traits, such as the ability to form a biofilm on a polystyrene surface, cell aggregation, and PIA production. The mutant could be complemented by transformation with the icaABC-carrying plasmid pCN27. Transfer of pCN27 into the heterologous host Staphylococcus carnosus led to the formation of large cell aggregates, the formation of a biofilm on a glass surface, and PIA expression. The nucleotide sequence of icaABC suggests that the three genes are organized in an operon and that they are co-transcribed from the mapped icaA promoter. IcaA contains four potential transmembrane helices, indicative of a membrane location. The deduced IcaA sequence shows similarity to those of polysaccharide-polymerizing enzymes, the most pronounced being with a Rhizobium meliloti N-acetylglucosaminyltransferase involved in lipo-chitin biosynthesis (22.5% overall identity and 37.4% overall similarity). This similarity suggests that IcaA has N-acetylglucosaminyltransferase activity in the formation of the beta-1, 6-linked N-acetyl-D-glucosaminyl polymer. IcaB is secreted into the medium and contains a typical signal peptide. IcaC is hydrophobic and contains six predicted transmembrane helices distributed over its entire length, typical for an integral membrane protein. Neither IcaB nor IcaC shares similarity with known proteins, and their function is unknown. Inactivation of icaA, icaB, or icaC in pCN27 led to the complete loss of the intercellular adhesion phenotype in S. carnosus, suggesting that all three genes are involved in intercellular adhesion, PIA expression, and translocation.

Lymphocyte subsets in the endometrium of genitally normal mares and mares susceptible to endometritis

The density and distribution of MHC Class II positive cells and subpopulations of lymphocytes were studied in the endometrium of genitally normal mares and mares susceptible to endometritis. In genitally normal mares, more MHC Class II positive cells were present in the epithelium and stratum compactum during oestrus than dioestrus. Significantly more CD4+ and CD8+ lymphocytes were present in the stratum compactum than in the stratum spongiosum. CD4+ lymphocytes were present in greater numbers than CD8+ lymphocytes in the stratum compactum but approximately equal numbers were present in the stratum spongiosum and in lymphoid aggregates. Occasional CD4+ and CD8+ cells were seen in the luminal and glandular epithelium. Infrequently, B cells were present in the endometrium and were not observed in the epithelium. Numbers of T and B cells did not appear to be affected by cycle stage. In mares with endometritis, the densities of CD4+, CD8+ and B cells were significantly increased. Large aggregates of lymphoid cells which contained approximately twice as many CD4+ cells as CD8+ cells were present in the endometrium of these mares and all 3 subclasses of lymphocyte were seen occasionally in luminal and glandular epithelium.

The effect of the type of closure on the gas composition of the headspace and the growth of GF 677 peach × almond rootstock cell suspension cultures

The influence of culture flask closures, i.e., cotton plugs and rubber and aluminum-foil caps, on headspace gas composition and growth of leaf petiole callus-derived GF 677 cell suspensions was comparatively tested. Oxygen concentration always remained comparable to that of the lab atmosphere and CO2 and C2H4 levels were slightly higher when flasks were closed with cotton plugs. By contrast, the gas environment markedly changed within 72 to 96 h in culture inside rubber and aluminum-capped flasks. Under rubber caps, CO2 increased up to 18%, with a net production of about 0.8 mmol CO2, oxygen decreased to 6% within 72 h, and ethylene accumulated up to 9 µl liter−1 after 96 h. When aluminum foil closures were used, C2H4 and CO2 increased over the first 72 h, reaching concentrations of about 6 µl liter−1 and 7% (0.3 mmol produced), respectively, and decreasing after 192 h to 0.1 µl−1 and 2%, respectively. The gaseous environment markedly affected cell growth. The exponential growth period of suspensions cultured in flasks closed with cotton plugs and aluminum foil caps started after about 72 h and the stationary phase after 240 h, the cell dry weight reaching its maximum at about threefold the initial weight. Large cell aggregates were found in flasks closed with cotton plugs, slight aggregation with aluminum closures, and no aggregates with rubber caps. However, hardly any growth, progressive browning of the suspensions, and the death of most cells occurred in rubber-capped flasks. A type of closure allowing low gas exchange rates, like aluminum caps, and frequent subcultures thus seems most conducive to rapid growing, more homogeneous GF 677 cell suspension cultures, and the prevention of aggregates, if undesired.