Sixth Passage Research Articles

IL-6 inhibits the proliferation of fibroblastic synovial cells from rheumatoid arthritis patients in the presence of soluble IL-6 receptor.

IL-6 and tumor necrosis factor (TNF)-alpha have been proven to play an important role in the development of rheumatoid arthritis (RA). It is well known that TNF-alpha induces IL-6 production from synovial cells as well as their proliferation. The effect of IL-6 on synovial cells, however, is not clear. An in vitrostudy was performed to determine the effect of IL-6 on the proliferation of synovial cells. Fibroblastic synovial cells isolated from the synovial tissues of eight RA patients were employed after the third to sixth passages. IL-6 in the presence of soluble IL-6 receptor (sIL-6R) inhibited the proliferation of synovial cells in a dose-dependent manner in seven cases without increasing the number of necrotic or apoptotic cells, while TNF-alpha increased synovial cell proliferation in all cases. The inhibitory effect of IL-6 was observed only in the presence of sIL-6R although small amounts of IL-6R were detected in these cells by RT-PCR analysis. However, anti-IL-6R or anti-gp130 mAb treatment increased spontaneous growth of synovial cells in all eight cases, suggesting that endogenous IL-6 and a small amount of IL-6R expressed in synovial cells suppressed their growth without exogenous IL-6 or sIL-6R. In addition, the IL-6-sIL-6R complex reduced the TNF-alpha-induced proliferation of synovial cells while TNF-alpha induced their IL-6 production. These data suggest that IL-6 may act as a negative feedback factor for TNF-alpha-induced synovial cell growth.

Diadenosine Polyphosphates and Atrial Natriuretic Peptide are Antiproliferative in Rat Mesangial Cells

Modulation of cell proliferation by vasoactive hormones and growth factors involves also changes in the activity of pH-regulatory transporters. In a preceeding paper (1) we examined the influence of such factors on cellular pH_i. Here the influence of the same factors, diadenosine polyphosphates (ApnA), atrial natriuretic peptide, the growth factor PDGF and the Ca²⁺-ATPase inhibitor thapsigargin, on proliferation of cultured rat mesangial cells was examined by quantification of [³H]-thymidine incorporation. Mesangial cells were synchronised and growth reduced (0.5% FCS for 24 h) before experiments were started. Incubation with Ap3A, Ap4A, Ap5A or Ap6A (all 10 μ) for 24 h all reduced cell proliferation by 30 to 45%. At 0.1 and 1 μM the effects of Ap4A, Ap5A and Ap6A did not reach significance. The antimitogenic effect of Ap5A was not significantly different when cells were incubated for 24, 48 or 72 h. In addition there was no significant difference between the antiproliferative effect of Ap5A in cells of the second, sixth or thirteenth passage. The growth factor PDGF-BB (0.25 nM) resulted in an approximately 3-fold increase in [³H]-thymidine incorporation. This increase in proliferation could be significantly reduced by coincubation with 10 μM Ap5A. The mitogenic effect of PDGF was completely abolished in the presence of the Ca²⁺-ATPase inhibitor thapsigargin (1 nM), which also significantly reduced basal cell proliferation by approximately 40%. Incubation of mesangial cells with 10 nM ANP for 24 h reduced basal [³H]-thymidine incorporation slightly by approximately 20% and decreased the PDGF-induced stimulation. The antimitogenic effects of these agonists is especially pronounced when cells are stimulated.

Activation of synovial fibroblasts in rheumatoid arthritis: lack of Expression of the tumour suppressor PTEN at sites of invasive growth and destruction.

PTEN is a novel tumour suppressor which exhibits tyrosine phosphatase activity as well as homology to the cytoskeletal proteins tensin and auxilin. Mutations of PTEN have been described in several human cancers and associated their invasiveness and metastatic properties. Although not malignant, rheumatoid arthritis synovial fibroblasts (RA-SF) exhibit certain tumour-like features such as attachment to cartilage and invasive growth. In the present study, we analyzed whether mutant transcripts of PTEN were present in RA-SF. In addition, we used in situ hybridization to study the expression of PTEN messenger (m)RNA in tissue samples of RA and normal individuals as well as in cultured RA-SF and in the severe combined immunodeficiency (SCID) mouse model of RA. Synovial tissue specimens were obtained from seven patients with RA and from two nonarthritic individuals. Total RNA was isolated from synovial fibroblasts and after first strand complementary (c)DNA synthesis, polymerase chain reaction (PCR) was performed to amplify a 1063 base pair PTEN fragment that encompassed the coding sequence of PTEN including the phosphatase domain and all mutation sites described so far. The PCR products were subcloned in Escherichia coli, and up to four clones were picked from each plate for automated sequencing. For in situ hybridization, digoxigenin-labelled PTEN-specific RNA probes were generated by in vitro transcription. For control in situ hybridization, a matrix metalloproteinase (MMP)-2-specific probe was prepared. To investigate the expression of PTEN in the absence of human macrophage or lymphocyte derived factors, we implanted RA-SF from three patients together with normal human cartilage under the renal capsule of SCID mice. After 60 days, mice were sacrificed, the implants removed and embedded into paraffin. PCR revealed the presence of the expected 1063 base pair PTEN fragment in all (9/9) cell cultures (Fig.1). No additional bands that could account for mutant PTEN variants were detected. Sequence analysis revealed 100% homology of all RA-derived PTEN fragments to those from normal SF as well as to the published GenBank sequence (accession number U93051). However, in situ hybridization demonstrated considerable differences in the expression of PTEN mRNA within the lining and the sublining layers of RA synovial membranes. As shown in Figure 2a, no staining was observed within the lining layer which has been demonstrated to mediate degradation of cartilage and bone in RA. In contrast, abundant expression of PTEN mRNA was found in the sublining of all RA synovial tissues (Figs 2a and b). Normal synovial specimens showed homogeneous staining fo PTEN within the thin synovial membrane (Fig. 2c). In situ hybridization using the sense probe gave no specific staining (Fig. 2d). We also performed in situ hybridization on four of the seven cultured RA-SF and followed one cell line from the first to the sixth passage. Interestingly, only 40% of cultured RA-SF expressed PTEN mRNA (Fig. 3A), and the proportion of PTEN expressing cells did not change throughout the passages. In contrast, control experiments using a specific RNA probe fo MMP-2 revealed mRNA expression by nearly all cultured cells (Fig. 3B). As seen before, implantation of RA-SF into the SCID mice showed considerable cartilage degradation. Interestingly, only negligible PTEN expression was found in those RA-SF aggressively invading the cartilage (Fig. 3c). In situ hybridization for MMP-2 showed abundant staining in these cells (Fig. 3d). Although this study found no evidence for mutations of PTEN in RA synovium, the observation that PTEN expression is lacking in the lining layer of RA synovium as well as in more that half of cultured RA-SF is of interest. It suggests that loss of PTEN function may not exclusively be caused by genetic alterations, yet at the same time links the low expression of PTEN to a phenotype of cells that have been shown to invade cartilage aggressively. It has been proposed that the tyrosine phosphatase activity of counteracting th actions o protein tyrosine kinases. As some studies have demonstrated an upregulation of tyrosine kinase activity in RA synovial cells, it might be speculated that the lack of PTEN expression in aggressive RA-SF contributes to the imbalance of tyrosine kinases and phosphatases in this disease. However, the extensive amino-terminal homology of the predicted protein to the cytoskeletal proteins tensin and auxilin suggests a complex regulatory function involving cellular adhesion molecules and phosphatase-mediated signalling. The tyrosine phosphatase TEP1 has been shown to be identical to the protein encoded by PTEN, and gene transcription of TEP1 has been demonstrated to be downregulated by transforming growth factor (TGF)-beta. Therefore, it could be hypothesized that TGF-beta might be responsible for the downregulation of PTEN. Low expression of PTEN may belong to the features that distinguish between the activated phenotype of RA-SF and the sublining, proliferating but nondestructive cells.

Mechanism of SNAP Potentiating Antiproliferative Effect of Calcitonin Gene-related Peptide in Cultured Vascular Smooth Muscle Cells

We previously showed that CGRP inhibits cell proliferation which correlates with an elevation of cAMP levels in rabbit aortic vascular smooth muscle cells (VSMCs). The present study determined the effects of S-nitroso-N-acetylpenicillamine (SNAP, a nitric oxide donor) on CGRP-induced antiproliferative effects and cellular mechanism in cultured rabbit aortic VSMCs. The cells (in fifth–sixth passage) were exposed to 2.5% fetal bovine serum for 24 h in the presence or absence of SNAP, hCGRP or both.3H-thymidine incorporation was used to measure DNA synthesis. The results showed that SNAP (60–100 μ m) significantly inhibited the proliferation and elevated cGMP levels in cultured rabbit aortic VSMCs. In combination, however, SNAP (30 μ m) potentiated hCGRP (10–100 n m)-induced antiproliferation. SNAP (30 μ m) and hCGRP (10–100 n m) or forskolin (10 μ m), an activator of adenylate cyclase, caused more than additive cAMP elevations, but not cGMP elevations, in these cells. Quazinone, an inhibitor of cGMP-inhibited-phosphodiesterase (cGI-PDE, PDE3), or SNAP plus quazinone caused a similar potentiation as SNAP of the hCGRP-induced elevations of cAMP levels. The data indicate that SNAP-induced potentiation of CGRP's effects likely involves inhibition of cGI-PDE, thus allowing enhanced accumulation of cAMP that mediates the antiproliferative effects of hCGRP in cultured rabbit aortic VSMCs.

CDCR With Buccal Mucosal Graft: Comparative and Histopathological Study

Conjunctivodacryocystorhinostomy (CDCR) is a procedure with a high functional success rate, on the other hand post-operative complications are frequent and tube replacement is often required. Therefore, new investigations were undertaken in order to relieve the undesired effects of the tube. Implantation of the Jones tube circumscribed with a buccal mucosal graft and subsequent removal of the tube when an epithelium lined track was formed has been reported in the literature in recent years. In this study efficacy and safety of this alternative technique was evaluated. CDCR with insertion of Jones tube surrounded with a buccal mucosal graft was performed on 14 patients (14 eyes) between September 1995 and March 1997 and results were evaluated. Results were compared with our previous series of traditional CDCR, involving 22 patients (22 eyes). Functional success was obtained in all cases with relief of epiphora after a mean follow-up of 13.7+/-5.7 months. In 11 cases, tubes were removed at the sixth post-operative month and passage was blocked in 9 cases. Subsequent surgical tube reinsertion was performed in those patients. Biopsy specimens were obtained in two cases during tube replacement and were examined histologically. Disarrangement of the basal epithelial cells and infiltration of the surrounding connective tissue with round cells were observed. These histopathological findings suggested a significant graft degeneration threatening the continuity of the passage. Tube displacement existed in two patients, and partial mucosal graft extrusion was apparent in one case during the period with the tube still inserted. Surgical tube replacement was required in only two cases to correct lateral migration of the tube. Incidence of complications and need for surgical tube replacement were found to be less than our previous series of 22 CDCR patients without buccal mucosa grafting. These findings suggest that CDCR with buccal mucosal graft is a promising new method for the treatment of epiphora because of its low incidence of complications during the period with the tube. However, tube removal at the sixth post-operative month is probably not indicated. Therefore, studies with larger series and longer follow-up should be undertaken to confirm the advantages of the technique.

Regulation of transmembrane signalling system during senescence of human epidermal keratinocytes.

Cultured normal human epidermal keratinocytes show an increase in doubling time with increasing passage number and finally the cells show growth arrest. This is known as senescence of cultured cells. However, the mechanisms governing senescence of cells remain to be clarified. Stimulation from outside the cell and the response to the stimulation are important essential initial events for cell function. Alteration of intracellular Ca2+ is one of the essential responses of cells to stimulation from outside. Thus we studied whether cultured normal human epidermal keratinocytes show a modulation of the alteration of intracellular Ca2+ during senescence. Epinephrine and histamine induced transient increases in intracellular Ca2+ in the third to eighth passages in normal human epidermal keratinocytes. With increase in the passage number the responsiveness (the number of responding cells per examined cells) decreased particularly beyond the sixth passage. The attenuation of the responses was more obvious with epinephrine than with histamine. All-trans-retinoic acid and 1,25-dihydroxyvitamin D3 did not augment the responsiveness to epinephrine of normal human epidermal keratinocytes. We expected that such an essential and immediate reaction would be confined to living cells, and that during senescence cells would show reduced responsiveness. Some of the changes during senescence of cultured keratinocytes may be due to the attenuation of the responsiveness to stimulation through the cell membrane.

Trypanosoma cruzi:Involvement of Intracellular Calcium in Multiplication and Differentiation

Lammel, E. M., Barbieri, M. A., Wilkowsky, S. E., Bertini, F., and Isola, E. L. D. 1996.Trypanosoma cruzi:Involvement of intracellular calcium in multiplication and differentiation.Experimental Parasitology83,240–249. The possible role of intracellular Ca2+level onTrypanosoma cruzidifferentiation was explored. The addition to epimastigotes of aTriatoma infestansintestinal homogenate, which that triggers off the differentiation to the infective metacyclic form, induced a sudden rise in [Ca2+]ifrom the basal value, 94 ± 28 to 584 ± 43 nmole/liter. This increase was not affected by the presence of EGTA in the medium. Trypsin-treated intestinal homogenate did not alter the [Ca2+]iof epimastigotes. Calmodulin inhibitors (Calmidazolium, Trifluoperazine, and Chlorpromazine) blocked differentiation. Although the calcium ionophore ionomycin increased [Ca2+]ito 342 ± 29 nmole/liter, it was unable to induce differentiation by itself. BAY K8644 and Methoxyverapamil (agonist and antagonist of Ca2+channels, respectively) were unable to affect [Ca2+]iby themselves, or when added to stimulated parasites, and did not exert a stimulatory or inhibitory effect on morphogenesis. BAPTA/AM, a Ca2+chelator, partially blocked the rise in [Ca2+]iand morphogenesis; this effect was reversed by ionomycin. The requirement of intracellular Ca2+on epimastigote multiplication was also evaluated. The addition of EGTA to the culture medium led to a decrease in epimastigote multiplication till it practically ceased in the sixth passage. When such parasites were transferred to LIT they partially recovered the growth rate. Parasites from passages III, IV, and V in the Ca2+-depleted medium maintained their basal [Ca2+]i, but when treated with the intestinal homogenate, the rise in [Ca2+]iwas abrogated. Accordingly, the differentiation percentages of such parasites dropped significantly compared with controls.

Construction and characterization of replication-competent simian immunodeficiency virus vectors that express gamma interferon

We report the construction and characterization of several replication-competent simian immunodeficiency virus (SIV) vectors with a deletion in the viral nef gene (SIV(delta nef)) that express gamma interferon (IFN-gamma). The expression of the cytokine gene was controlled either by the simian virus 40 early promoter or by the SIV 5' long terminal repeat regulatory sequences, utilizing the nef gene splice signals. To enhance the expression of IFN-gamma, the two in-frame nef start codons were mutated without altering the Env amino acid sequence (SIV(HyIFN)). Plasmids containing full-length proviral genomes were used to obtain high-titer stocks of each recombinant virus in cell cultures. Expression of IFN-gamma by SIV(HyIFN) reached levels as high as 10(6) U/ml after 11 days in culture. The IFN-gamma gene was unstable and sustained deletions after serial passage of SIV(delta nef) vectors in CEM-X-174 cells. The degree of instability appears to depend on size and orientation of the insert and the expression of IFN-gamma. Only one virus, SIV(HyIFN), expressed detectable levels of IFN-gamma up to the sixth passage. Prospects for the use of IFN-gamma and other lymphokines to enhance the safety and efficacy of live attenuated vaccines are discussed.

A new experimental model for studying the response of periodontal ligament cells to hydrostatic pressure

An apparatus was developed to apply positive or negative hydrostatic pressure dynamically to periodontal ligament (PDL) cells in vitro. The objective of this investigation was to construct this apparatus and to determine its effects on PDL cells. Human PDL cells were collected from freshly extracted premolars. At the sixth passage, the cells were mechanically stimulated by this apparatus at different magnitudes of continuous positive or negative hydrostatic pressures (PHP or NHP, respectively). The application of PHP between 0.3 and 30 gm/cm 2 significantly enhanced prostaglandin E (PGE) production and intracellular cyclic AMP (cAMP) of the cells. In contrast, perturbation by NHP significantly decreased PGE production and intracellular level of cAMP. Proliferation rate increased significantly at 24 and 48 hours due to stimulation of these cells with −30 gm/cm 2 of NHP. Challenging these cells with +30 gm/cm 2 of PHP significantly decreased the proliferation rate of these cells at 24 and 48 hours. Stimulation by PHP between +30 to +600 gm/cm 2 increased cell length and width and appeared to increase surface area attachment to the bottom of the culture dishes. In contrast, NHP (between −30 and −600 gm/cm 2) decreased these dimensions and appeared to reduce the surface area of attachment. These results indicate that this type of mechanical perturbation of PDL cells produces physiologic responses and is not detrimental to their vitality. (A M J ORTHOD D ENTOFAC O RTHOP 1995;108:402-9.)

Preferential selection of specific rotavirus gene segments in coinfection and multiple passages with reassortant viruses and their parental strain

We previously reported non-random selections of human rotavirus (HRV) Wa genes 2 and 5 in reassortant formation between HRV strains Wa and HN126 under selection pressure with neutralizing monoclonal antibodies. In order to study whether or not these genes are preferentially selected in the genetic background of a parental strain HN126 in vitro without selection pressures, coinfection and multiple passage experiments were performed between HN126 and one of three reassortants, C1, C1T and C1F; C1 possessed genes 2 and 5 derived from Wa and the other genes derived from HN126, while C1T and C1F were single gene reassortants having Wa gene 2 or Wa gene 5 in the genetic background of HN126, respectively. When MA-104 cells were coinfected with the same infectious units of HN126 and C1, Wa genes 2 and 5 of reassortant C1 became predominant within 10 repeated passages, although Wa gene 5 was selected more preferably than Wa gene 2. Similar results were obtained under different experimental conditions in which different doses of parental strains or different type of cells were used. Also, in coinfections of MA-104 cells with HN126 and C1T, or HN126 and C1F, Wa gene 2 or Wa gene 5 became predominant at the sixth passage. Analysis of viral growth curves indicated that two reassortants, C1 and C1F, replicated to a titre higher than HN126, while no difference in viral growth was observed between C1T and HN126. These results indicated that in the genetic background of HN126, Wa gene 5 might provide viruses with a growth advantage compared with its HN126 counterpart, while Wa gene 2 might be preferentially selected into reassortant clones through its greater functional capacity for assortment during viral replication.

Binding, coupling, and mRNA subtype heterogeneity of α1-adrenergic receptors in cultured human RPE

In retinal pigment epithelium, apically applied epinephrine changes the conductance of specific ions which subsequently affects the membrane voltage and stimulates transepithelial fluid transport. In this investigation, myo-[3H]inositol radiotracer studies, radioligand binding with [125I]HEAT, and ribonuclease protection assays were performed to examine the coupling of this receptor to phosphoinositide hydrolysis and the specific mRNA subtypes expressed in cultured human retinal pigment epithelial cells. After labeling second to sixth passage cells with 3-muCi myo-[3H]inositol for 24 hr, epinephrine caused a dose- and time-dependent increase in [3H]inositol phosphate products (EC50 of 0.7 microM). This stimulation was antagonized by prazosin but not by propranolol. The effect of epinephrine was potentiated by the presence of the monoamine oxidase inhibitor, pargyline (10 microM). Pertussis toxin (1 microgram per well) attenuated the stimulatory effect of epinephrine. In the radioligand binding assays, [125I]HEAT binding sites varied among different cell lines, with a range of 44 to 200 fmol (mg protein)-1. Using a ribonuclease protection assay, alpha 1D and alpha 1B, but not alpha 1C, adrenergic mRNA subtypes were detected in cultured human cells. Collectively, these results show that the catecholamines act on a potentially heterogeneous population of alpha 1-adrenergic receptors coupled to phospholipase C by a pertussis toxin-sensitive G protein.

Consistent responses of the human vascular smooth muscle cell in culture: Implications for restenosis

Purpose: The mechanisms whereby restenoses occur at discrete sites within the vasculature remain uncertain. We have recently reported that vascular smooth muscle cells (VSMC) derived from patients with graft stenoses are resistant to growth inhibition by heparin. In this study, we have examined whether VSMC proliferation rates and responses to inhibition by heparin vary according to the individual or the anatomic site of origin.Methods: Long saphenous veins from seven patients were divided into proximal, middle, and distal portions, and VSMC were cultured separately from each. VSMC proliferation in response to 15% fetal calf serum ± 100 μg/ml heparin was measured by counting triplicate samples at 0, 3, 7, 10, and 14 days. This experiment was repeated from the second to the sixth passage (n = 6) and for artery and vein pairs derived from four additional patients.Results: Differences between vein segment cultures of individual veins were found not to differ significantly from experimental error for either proliferation or heparin inhibition and were not altered by repeated passage (ANOVA). There were, however, significant differences in sensitivity to heparin inhibition between patients (p = 0.02) (ANOVA). There were no significant differences between paired samples of artery and vein for either proliferation or heparin inhibition (Mann-Whitney test).Conclusions: VSMC growth characteristics reflect the individual patient and are maintained in cell culture.

Drug-resistant schistosomiasis: resistance to praziquantel and oxamniquine induced in Schistosoma mansoni in mice is drug specific.

Schistosoma mansoni infections in mice were treated with subcurative multiple doses of either praziquantel (PZQ) or oxamniquine (OX). With an early exception, the drug treatments commenced when the worms were adult, but before the infections had become fully patent, and the eggs subsequently produced by worms that had survived the drug treatments were used to infect snails. Six or seven drug-treated passages of S. mansoni in mice were completed for each of the drugs, with the amount of drug administered to the infected mice generally being increased with each passage. Eighty percent of the worms of the sixth passage selected for PZQ resistance survived three doses of 300 mg/kg of PZQ given between days 28 and 37 after infection, and 93% of those of the seventh passage survived the same drug dose. In contrast, only 13% of worms of the sixth PZQ-selected passage survived three doses of 200mg/kg of OX given during the same period after infection. Only 11% or fewer worms derived from S. mansoni infections that had not been subjected to any drug pressure survived the 3 x 300 mg/kg PZQ treatments. Worms selected for OX resistance over six passages were completely resistant to three doses of 200 mg/kg, but only 26% survived three doses of 300 mg/kg of PZQ. Therefore, the results indicate that S. mansoni subjected to drug pressure may develop resistance to schistosomicidal drugs over the course of relatively few passages, but that cross-resistance between PZQ and OX does not occur. This is the first demonstration of drug resistance to PZQ, the current drug of choice for human schistosomiasis.

Culture of renal arteriolar smooth muscle cells. Mitogenic responses to angiotensin II.

We cultured smooth muscle cells from rat renal preglomerular arterioles by injecting a suspension of iron oxide into the left ventricle, separating the arterioles magnetically, and growing cells from explants. In passaged cultures we ascertained vascular smooth muscle purity of > 98% by morphology; contraction to norepinephrine and angiotensin; positive immunofluorescence staining through the sixth passage with monoclonal antibodies to smooth muscle-specific alpha- and gamma-isoactins, myosin, and desmin; and the absence of von Willebrand factor. Angiotensin II (10(-12)-10(-5) M) induced dose-dependent DNA synthesis and proliferation of subcultured (three times) arteriolar smooth muscle cells from a growth-arrested state (p < 0.01). Angiotensin II (10(-5) M) also induced the cells to express c-fos mRNA. We find no previous report of culture of smooth muscle cells from renal preglomerular arterioles. Our findings also provide evidence that angiotensin II is mitogenic to arteriolar muscle cells and thus may be involved in their hyperplasia accompanying hypertension.

Demonstration of the Genetic Stability of a Mycoplasma gallisepticum Strain Following in vivo Passage

A Mycoplasma gallisepticum strain designated 6/85 (MGI) exhibiting reduced virulence for both chickens and turkeys was sequentially passaged 10 times in each species. DNA extracted from organisms before passage and those isolated after the third, sixth, and 10th passages was studied by restriction endonuclease DNA analysis using BamHI, BglII, EcoRI, HindIII, and PstI endonucleases. The virulent-type strain designated S6 was used as a comparison. Comparison of DNA fragment patterns of MGI and S6 strains showed distinct differences, although some similarities were evident. Passage of the strain in vivo did not affect DNA fragment patterns of the MGI strain. Electrophoretic protein patterns produced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed very similar band patterns in both the MGI and S6 strains. The most notable differences were seen in bands located in the molecular-mass regions of approximately 46.5, 50-54, 58-64, and 105-140 kilodaltons. Alteration of band pattern profiles following in vivo passage of the MGI strain was apparent in a single band at approximately 86 kilodaltons that appeared to stain more intensely following passage.

In Vitro Micropropagation of Delphinium malabaricum (Huth) Munz.—a Rare Species*

A micropropagation technique was developed for Delphinium malabaricum using nodes from inflorescence stalks. Maximum shoot proliferation was obtained on Murashige and Skoog's medium supplemented with 2-iP (10 mg1 -1 ) and inositol (100 mg1 -1 ). From the sixth passage onwards, shoots could be multiplied by omission of inositol and reduction of 2-iP (0.5 mb1 -1 ) concentration. Best rooting response was obtained with a 24-h pulse treatment of shoots with 0.5 mg1 -1 IBA in the dark, transfer of treated shoots to hormone-free half-strength MS medium and incubation under 24-h light. Regenerated plants were established successfully in the field (...)

Interactive Effects Between Cytokines on PGE Production by Human Periodontal Ligament Fibroblasts in vitro

Mononuclear cell production of cytokines that stimulate fibroblast production of prostaglandin E (PGE) is an important mechanism by which mononuclear cells regulate fibroblast function. The objective of this investigation was to determine the effects of the cytokines interleukin 1 beta (IL-1 beta), interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma), alone or in paired combinations, on PGE production by near-confluent human periodontal ligament (PDL) fibroblasts in vitro. Premolars extracted in the course of orthodontic treatment were used for this study. Fibroblast cultures, free of epithelial cells, were obtained after the fourth subculture by the use of accurately-timed trypsin treatment. Cells in the fourth to sixth passage, incubated in DMEM supplemented with 10% equine serum, were used for these experiments. Cells (1 x 10(5)) were seeded in 12- x -75-mm tissue culture tubes and incubated with various doses of IL-1 beta, IL-1 alpha, TNF-alpha, and IFN-gamma, alone or in specific combinations, for 15 min, two, 12, 24, and 72 h. PGE concentrations in the media were measured by radio-immunoassay. The results showed that human PDL fibroblasts responded to the administration of cytokines by an elevation in the synthesis of PGE in a dose- and time-related fashion. The increase in PGE production was inhibited by the addition of indomethacin. The interactions between these cytokines varied in degree, depending on the particular combinations of cytokines. In addition, the administration of cytokine combinations was found to be additive, synergistic, subtractive, or suppressive on the production of PGE by PDL fibroblasts, depending on the duration of incubation. These experiments demonstrate the importance of the consideration of the interplay between cytokines produced by mononuclear cells on the mechanisms that regulate the functions of PDL fibroblasts.

Assessment of Human Genitourinary Tumors and Chemosensitivity Testing In 3-Dimensional Collagen Gel Culture

A recently described collagen gel culture technique has been modified to evaluate the growth characteristics and chemosensitivity patterns of genitourinary neoplasms. Fresh human surgical explants incorporated radiolabeled DNA precursors ((H3)thymidine or deoxyuridine) in 97% of 38 patient specimens (18/19 bladder, 3/3 prostate, 2/2 testis, 13/14 renal), after being maintained for three to 13 weeks/passage, with several specimens reaching their sixth passage (20 months). Control cellular DNA incorporation ranged from five to 90% (#cells labeled/#cells evaluated), with median labeling for bladder 30%, prostate 80%, testis 90%, and renal 80%. Original histopathologic classification was maintained in all cases. Tumor volume and glucose consumption were other measurable parameters. Seventy-three surgical specimen cultures were treated with chemotherapeutic agents after a minimum of four weeks in culture. Single agent exposures were 24hours at lx and 10X reported peak plasma concentrations. Combination agents were sequenced as in current clinical protocol for bladder tumors and fourteen day continuous fluorodeoxyuridine (FdURD) exposure for renal tumors. Sensitivity was found in 1/2 prostate and 1/10 renal tumors to Adriamycin, 8/15 bladder and 1/2 testis tumors to cisplatin, 11/28 renal tumors to FdURD and 6/ 16 bladder tumors to MVAC combination chemotherapy.This culture system offers the advantages of in vivo-like solid tumor growth, a high culture success rate, longevity in culture, maintenance of the primary histopathology and reproducible chemosensitivity response. (J. Urol., 143:1041-1045, 1990)

A new cell line (8701-BC) from primary ductal infiltrating carcinoma of human breast

A cell line, designated 8701-BC, was established in culture from tissue fragments of primary ductal infiltrating carcinoma of human breast. The cell cultures after the sixth passage were devoid of contaminating fibroblasts as judged by the positive staining of all cells with the specific epithelial cell markers carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA) and cytokeratin 8. The epithelial nature of these cells was confirmed by ultrastructural analyses which demonstrated the retention of specific structural properties characteristic of the original tumour. The cells possessed an abnormal karyotype with 55-60 chromosomes per cell with numerous rearrangements. They do not express HLA antigens and the c-myc gene was not amplified. The 8701-BC cells have a doubling time of approx. 29h and have been maintained in culture for more than 100 passages. These properties suggest the establishment of a human neoplastic cell line which, with its ability to produce homotrimer collagen in vitro, will provide a useful model system for the study of tumour cell:stromal matrix interactions.

Enhancement of Campylobacter jejuni Virulence by Serial Passage in Chicks

Four isolates of Campylobacter jejuni were studied to determine changes in virulence following six serial passages in chicks. Chicks that received invasive isolates exhibited diarrhea and depressed weight gain. Immature mice were used to assess virulence of the passaged isolates of C. jejuni. Nine-day-old mice infected with passaged isolates showed lethargy, dehydration, depression, decreased weight gain, and occult blood in feces. Mouse pups inoculated with the third and sixth chick passage levels of an invasive isolate showed significant depression in mean daily weight gain and elevated mortality compared with controls and subjects inoculated with unpassaged isolates. This study demonstrated enhancement of virulence in a C. jejuni isolate following chick passage. In contrast, three other passaged isolates failed to show any consistent increase in virulence.