Cytotoxic effect of diphtheria toxin used alone or in combination with other agents on human renal cell carcinoma cell lines

Treatment of renal cell carcinoma (RCC) by conventional chemotherapy and immunotherapy has resulted in minimal remissions. Alternative forms of therapy are therefore being sought. The present study investigated the sensitivity of RCC cell lines to several toxins used alone and in combination with other agents. RCC lines were relatively sensitive to the direct cytotoxic effect of diphtheria toxin (DTX), Pseudomonas aeruginosa exotosin A (PEA) and ricin. Furthermore, DTX in combination with tumor necrosis factor- (TNF-) resulted in synergistic cytotoxic activity. The mechanism of synergy was examined. A possible mechanism of resistance to TNF- in tumor cells is the expression of TNF- mRNA or protein. R11 cells did not constitutively express mRNA for TNF-, however, treatment of R11 cells with TNF- induced the expression of TNF- mRNA. When DTX was used in combination with TNF-, the level of TNF- mRNA induced by TNF- was markedly reduced. These studies suggest that DTX in combination with TNF- can overcome the resistance of RCC lines and that the marked downregulation of TNF- mRNA by DTX may play a role in the enhanced cytotoxicity seen with the combination of DTX and TNF-. Furthermore, the combination treatment might also potentiate the antitumor host responses. The implications of these findings in clinical therapy are discussed.     

Receptor function studies in specimens from the proximal human urethra obtained by transurethral resection

Summary During transurethral resection (TUR) for prostatic hyperplasia, specimens were taken from the proximal urethra. Muscle strips thus obtained were mounted in an organ bath and muscle contraction was induced by adding increasing concentrations of noradrenaline (NA), methoxamine (₁-agonist) and clonidine (₂-agonist). NA and methoxamine induced a dose-dependent muscle contraction, but clonidine had no effect. The influence of prazosin (₁-antagonist) and yohimbine (₂-antagonist) on the NA-induced muscle contraction was also evaluated. Both antagonists had an inhibitory effect,which was much more potent with prazosin. The specimens taken during TUR were found to be suitable for in vitro receptor function studies. The -adrenergic receptor function in the proximal human urethra was found to be mainly of the -type.     

Lysosomal localization of secretory prostatic acid phosphatase in human hyperplastic prostate epithelium

Summary The ultrastructural localization of secretory prostatic acid phosphatase (PAP) in human benign prostate tissue was accomplished using the immunogold technique on ultrathin Lowicryl sections. Polyclonal antibodies directed against secretory PAP (MW 50 kD) and the lysosomal enzymes -glucosidase and -galactosidase as well as an antiserum dircted against prostatic antigen (PA) were used. PAP was found in secretory vacuoles of columnar secretory epithelial cells. In addition, double labeling experiments revealed that secretory PAP was also localized in electrondense organelles of columnar epithelial cells containing -glucosidase and -galactosidase. PA was exclusively found in secretory vacuoles of columnar secretory epithelial cells. The results demonstrate the presence of secretory PAP within functional lysosomes and secretory vacuoles of the prostatic columnar epithelial cells and the absence of such PAP-containing lysosomes in the basal cells of the prostatic acini.     

Interferon-β Is More Potent Than Interferon-α in Inhibition of Human Hepatocellular Carcinoma Cell Growth When Used Alone and in Combination With Anticancer Drugs

Background:The prognosis of advanced hepatocellular carcinoma (HCC) is extremely poor, but promising effects of chemotherapies combined with interferon (IFN) have been reported.Methods:To develop more effective combination therapies for HCC, we compared the antiproliferative effects of IFN- and IFN- in combination with various cytotoxic drugs on hepatoma cell lines using MTT assay and isobologram analysis.Results:IFN- was more potent than IFN- in inhibiting the cell growth of all cell lines (P < .05, two-way ANOVA). PLC/PRF/5 was more sensitive to either IFN, than HLE and HuH7. Cell growth of all cell lines was inhibited in a dose-dependent manner by 5-fluorouracil (5-FU), cisplatin (CDDP), and doxorubicin (DOX), but the sensitivities of these cells were considerably different. As for IFN-, synergistic effects were observed when combined with 5-FU and DOX on PLC/PRF/5 cells only, whereas IFN- showed synergistic effects with 5-FU and CDDP on HuH7 and PLC/PRF/5 cell lines.Conclusion:The spectra of the antiproliferative activity and synergistic effect of IFN- when combined with anticancer drugs are more potent than those of IFN-. Combinations of IFN- and anticancer drugs may provide a better treatment of HCC when combinations with IFN- are ineffective.Presented at the 56th Annual Cancer Symposium of the Society of Surgical Oncology, Los Angeles, California, March 5–9, 2003.     

In Vivo Effect of Tumor Necrosis Factor Alpha on Wound Angiogenesis and Epithelialization

Abstract Background: The role of tumor necrosis factor alpha (TNF-) in wound healing is unclear and the results are contradictory. In vivo, TNF- induces vessel growth, an important step in promoting wound healing. However, a reduced amount of collagen, hydroxyproline, and granulation tissue was found after TNF- treatment. It is also unknown if this is a direct effect, by influencing cells involved in wound healing, or an indirect effect due to a negative or positive effect on cells such as macrophages. Material and Methods: The current study was undertaken to test the effect of TNF- on wound epithelialization and neovascularization in vivo. In the first experiment, standardized full-thickness dermal wounds (2.25 mm diameter, 0.125 mm depth) were created on the dorsum of the ears of male hairless mice. The wounds were treated either with TNF- (100 ng/ml, 1 µg/ml, 5 µg/ml; n = 10 per group), monoclonal TNF- antibody (10 µg/ml; n = 10), or vehicle (n = 10). Wound epithelialization and neovascularization were analyzed every 3rd day by intravital microscopy until complete healing.In a second experiment, the same wound model was used, but in order to impair the healing process, macrophages were depleted. To reduce macrophages, two out of four groups (n = 10 per group) were pretreated with iota-carrageenan (MR groups), and the other two groups received only saline (N groups). One N group and one MR group were treated with TNF- (1 µg/ml). The other N group and MR group received vehicle only (carboxymethylcellulose). Using intravital microscopy and computerized planimetry, wound epithelialization and neovascularization were measured every 3rd day until complete healing. Immunohistochemistry was performed to detect TNF-, macrophages, fibronectin, and vitronectin receptors. Results: In the first experiment the wounds treated with 1 µg/ml healed significantly earlier than controls (13.9 ± 2 vs. 17.3 ± 2.8 days, respectively; p < 0.05). Epithelialization in the antibody group was significantly slower compared to controls (20.1 ± 2 days; p < 0.05). Neovascularization was significantly enhanced in the group treated with 1 µg/ml TNF- (p < 0.05). In the second experiment the wounds treated with TNF- were significantly earlier epithelialized (13.1 ± 0.6) and vascularized (16.0 ± 0.5) compared to controls (16.8 ± 0.4 vs. 17.6 ± 0.5; p < 0.05). Wound closure was significantly delayed in the MR group treated with vehicle only (20.4 ± 1) and equal to controls in the MR group treated with TNF- (16.8 ± 0.6). Conclusion: The results demonstrate the TNF- accelerates wound epithelialization and neovascularization in this in vivo model. TNF- is able to compensate for the negative effect of macrophage reduction and seems to have a direct effect on the wound-healing process.     

Collagen distribution in human membranous glomerulonephritis

In membranous glomerulonephritis (MGN), thickening of the glomerular basement membrane (GBM) is partly due to the accumulation of basement membrane material between and around immune deposits located on the epithelial aspect of the GBM. We investigated the distribution of type IV collagen chains (1/2, 3, 4, 5, 6) and of types I, III, V, and VI collagen in the glomeruli from 16 patients, by indirect immunofluorescence in 13 and the high-resolution immunogold technique in 6. No changes were detected in stage I MGN. The spiky projections of the GBM in stage II MGN and the basement membrane layers encircling immune deposits in stage III contained the 3, 4, and 5 chains of type IV collagen. In contrast, the 1/2 chains of type IV, as well as type VI collagen accumulated in the subendothelial aspect of the GBM. No significant staining for types I, III, and V collagens or for the 6 chain of type IV collagen was detected. The results show that, as in the normal glomeruli, the different chains of type IV collagen are not co-distributed in the glomerular extracellular matrix in MGN. They also indicate that type IV collagen chains and type VI collagen play an important role in the thickening of the GBM in human MGN.     

Glomerular Expression of α-Smooth Muscle Actin (α-SMA) in Idiopathic Mesangiocapillary Glomerulonephritis Type I. A Quantitative Study

Eighteen renal biopsy specimens from patients with mesangiocapillary glomerulonephritis type I (MCGN-I) evaluated by both light and electron microscopy as well as immunofluorescence microscopy and whose full clinical data were available were examined quantitatively. As control 10 biopsy specimens of kidneys removed because of trauma were used. Morphometric investigations were performed by means of a computer image analysis system to evaluate the glomerular expression of -SMA in MCGN-I, as well as to determine whether this parameter could correlate with quantitatively analysed glomerular cells and mesangial areas. Another purpose of this study was to verify if the expression of -SMA correlated with the intensity of glomerular leukocyte infiltrates in this glomerulopathy. The morphometric study revealed that mean values of the expression of -SMA, total glomerular cells per total glomerular area, mesangium (% of total glomerular area), CD 45RB+, CD 43+, CD 20+ and CD 68+ cells were in MCGN-I patients increased in comparison with normal controls. Moreover, in the MCGN-I group, but not in controls, significant positive correlation existed between the glomerular expression of -SMA and total glomerular cells per total glomerular area, mesangium (% of total glomerular area), as well as CD 45RB+ cells and CD 68+ cells. Significant positive correlation between -SMA staining and total glomerular cells and mesangial areas suggested that increased -SMA expression might be in MCGN-I an indicator of mesangial cell activation and mesangial matrix production. The significant positive correlation between the glomerular expression of -SMA and glomerular CD 68+ cells requires further investigations to elucidate whether monocytes/macrophages play a role in the process of inducing myofibroblast phenotype in mesangial cells.     

Natural human IgG inhibits the production of tumor necrosis factor-α and interleukin-1α through the Fc portion

The overproduction of cytokines such as the tumor necrosis factor- (TNF-) and interleukin-1 (IL-1) may cause further deterioration in the already critical condition of patients with shock, sepsis, and acute inflammation. The effectiveness of infusion therapy of natural human IgG to such patients is suggested to depend partly upon the inhibition of the productivity of these cytokines. In this study, we investigated the modulation effects of IgG and its fragments on the production of TNF- and IL-1, on human peripheral blood mononuclear cells (PBMC). The production of TNF- and IL-1 was found to be dose-dependently inhibited by IgG when stimulated by lipopolysaccharide (LPS), phytohemagglutinin (PHA), concanavalin A (Con A), and interleukin-2 (IL-2). However, no inhibition was seen when stimulated by phorbormyristate acetate (PMA). The F(ab)₂ fragment showed enhancing effects on cytokine production by LPS, while the Fc fragment showed not as much inhibitory effect as whole intact IgG. IgG showed no direct cytotoxic effect on PBMC. These data suggest that natural human IgG inhibits TNF- and IL-1 production by PBMC through the Fc portion. The results of this study led us to conclude that whole intact IgG may be the best form of therapeutic delivery.     

Comparison of adrenoceptor subtype expression in porcine and human bladder and prostate

We have quantified and characterized ₁-, ₂-and -adrenoceptor subtypes in porcine bladder detrusor and bladder neck, human bladder detrusor, and porcine and human prostate. ₁-, ₂- and -adrenoceptor were identified in radioligand binding studies using [³H]prazosin, [³H]RX 821002 and [¹²⁵I]iodocyanopindolol, respectively, as the radioligands. In porcine male and female detrusor and bladder neck and male prostate, adrenoceptors were detected in the order of abundance > _{2 1} (not detectable), with no major differences between the sexes or between detrusor and bladder neck. In human detrusor and prostate the order of abundance was > _{2 1} (not detectable) and ₁ > ₂. respectively. The ₂-adrenoceptors in all tissues were homogeneously of the _2A-subtype as evidenced by competition binding studies with yohimbine, prazosin, ARC 239 and oxymetazoline. The -adrenoceptors represented a mixed population with a dominance of the ₂-subtype in all tissues as demonstrated by competition binding with ICI 118,551 and CGP 20,712A. We conclude that pigs may be a suitable model for studies of detrusor function with respect to adrenoceptor expression. They may be less suitable for studies of bladder neck or prostate function.     

Cytotoxic effects of alpha- and gamma-interferon and tumor necrosis factor in human bladder tumor cell lines

We investigated the activity of alpha-interferon (-IFN), gamma-interferon (-IFN) and tumor necrosis factor-alpha (TNF-) in a panel of ten human bladder tumor cell lines. All cytokines were tested at concentrations of 100–10000 U/ml in a clonogenic assay system. We found that -IFN was active against five of the ten lines while -IFN was only active against one line. TNF was active against five of the ten lines. Maximum synergisms were obtained between the -IFN and TNF, occurring in nine of the ten cell lines. We conclude that -IFN and TNF are active as single agents and synergistic when used together in vitro in human bladder tumor cell lines.     

Role of endogenous interferon gamma in murine tumor growth and tumor necrosis factor alpha antitumor efficacy

Background: The anticancer role of tumor necrosis factor-alpha (TNF-) has been limited by toxicity. These experiments evaluate blocking endogenous interferon-gamma (IFN-) activity to abrogate TNF- toxicity. Methods: C57Bl/6 mice bearing MCA 105 tumor were treated with TNF- and anti-IFN- antibody (Ab) to evaluate the effect on the acute lethality of TNF- and their efficacy as evaluated by tumor growth rate, tumor histology, and survival. Results: Anti-IFN- Ab decreased TNF- lethality. Anti-IFN- Ab alone increased tumor growth significantly more than did nonimmune IgG (p₂<0.0001). Tumor-bearing mice that received nonimmune IgG and TNF- had slower tumor growth (p₂<0.02) and a trend toward improved survival (p=0.07) compared with saline-treated controls. Anti-IFN- Ab abrogated the antitumor effect of TNF-, prevented acute tumor necrosis histologically, and resulted in tumor growth rate and host survival similar to that of controls. The findings in mice that received anti-IFN- Ab and high-dose TNF- were comparable with those in mice that received a lower, equitoxic dose of TNF- alone. Conclusions: Blocking endogenous IFN- accelerates tumor growth in this model and partially abrogates the toxic and antitumor activity of exogenous TNF- equally. This suggests that blocking endogenous IFN- activity is not a useful strategy for limiting TNF- treatment toxicity.Presented in part at the 45th Annual Cancer Symposium of The Society of Surgical Oncology, New York, New York, March 15–18, 1992.     

Enhanced gene expression of transforming growth factor-α and c-met in rat urinary bladder cancer

To investigate the roles of growth factors in bladder cancer, changes in the expression of messenger RNAs (mRNAs) for several growth factors and their receptors were examined during rat bladder carcinogenesis induced with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Northern blot analysis showed that the contents of mRNAs for transforming growth factor- (TGF-) and c-met/hepatocyte growth factor (HGF) receptor increased with BBN treatment. Epidermal growth factor (EGF) receptor mRNA was hardly affected by the treatment; while mRNA for fibroblast growth factor (FGF) receptor 1 and transforming growth factor- (TGF-) type II receptor decreased with BBN treatment. A rat bladder tumor cell line, NBT-II, expressed both TGF- and c-met mRNAs, and HGF showed apparent scattering and growth-stimulating effects on the cells. These results indicate the possibility that TGF- produced by a bladder cancer, in addition to urinary EGF, plays a role in the development of bladder cancer, and that enhanced cell motility due to activation of the c-met/HGF receptor participates in the invasion and metastasis of the cancer cells.     

Stimulation of intercellular adhesion molecule-1 (ICAM-1) antigen expression and shedding by interferon-γ and phorbol ester in human renal carcinoma cell cultures: relation to peripheral blood mononuclear cell adhesion

In the present study we investigated the effect of interferon- (IFN-) and phorbol-12-myristate 13 acetate (PMA) on intercellular adhesion molecule-1 (ICAM-1) antigen expression and shedding in human renal carcinoma cell cultures. We also examined the functional consequences of ICAM-1 antigen expression and soluble ICAM-1 molecules on the adhesion of peripheral blood mononuclear cells (PBMC). Incubation of the human renal carcinoma cell line CaKi-1 with IFN- or PMA enhanced ICAM-1 antigen expression. The calcium ionophore, 4-bromo-calcium ionophore A23187 (Bromo-A23187) significantly enhanced the IFN- and PMA effect. Soluble ICAM-1 (sICAM-1) was detected in the supernatants of stimulated but not unstimulated cultures, and correlated significantly with cellular expression. Using ⁵¹Cr-labelled peripheral blood mononuclear cells in a cell adhesion assay, we demonstrated increased adhesion in IFN--treated CaKi-1 cultures, which was augmented by Bromo-A23187. This adhesion was blocked by preincubation of CaKi-1 cells with monoclonal antibody against ICAM-1 or by preincubation of PBMC with either monoclonal antibody against leucocyte function associated antigen-1 (LFA-1), a major receptor for ICAM-1, supernatants from treated cultures or purified sICAM-1 molecules. Thus, shedding of ICAM-1 may play a role during the escape from immunosurveillance by renal carcinoma cells.     

Renal α-adrenergic receptors and genetic hypertension

The hypothesis has been proposed that an increase in the number of renal -adrenergic receptors may contribute to the pathogenesis of genetic hypertension. Herein we review recent findings regarding expression of renal ₁ (_{1 a},_{1 b})- and ₂ (_{2 a},_{2 b})-adregenergic subtypes and we provide an updated revision of the above-stated hypothesis. Enhancement in receptor number or in post-receptor components responsible for ₁- and ₂-adregenergic mediated sodium reabsorption in proximal tubule may contribute to sodium retention and an elevation in blood pressure. Perhaps such changes contribute to the increase in blood pressure in genetically determined hypertension in humans, although direct tests of this notion have not yet been performed.     

Differentiation of pancreatic ductal carcinoma cells associated with selective expression of protein kinase C isoforms

Background: The signal transduction pathways important in regulating the growth and differentiation of malignant cells are poorly understood. Recent evidence has implicated activation of the protein kinase C (PKC) family of signaling proteins in pancreatic carcinoma during cytokine-induced cytostasis and differentiation. Methods: A human pancreatic adenocarcinoma (HPAC) cell line was exposed to tumor necrosis factor- (TNF-; 40 ng/ml) for 6 days. Cytostasis and viability were confirmed by daily MTT [(3(4,5)-dimethyl-thiazol-2-yl) 2,5-diphenyl-tetrazolium bromide] and trypan exclusion assay. Protein fractions were isolated daily and subjected to immunoblot analysis for the normal (terminally differentiated) pancreatic ductal cell marker carbonic anhydrase II (CA II) as well as specific PKC isoforms (, , , , and). Results: Growth arrest occurred in HPAC cells after exposure to TNF- for 48 h, with viability maintained above 90% throughout the 6-day time course. CA II immunoreactivity was not detected in untreated controls but appeared after 2 days of TNF- exposure, peaking on day 6. Concurrently, TNF- induced the selective downregulation of PKC-, whereas PKC- levels increased. PKC- and PKC- immunoreactivity did not change. The atypical PKC- isoform developed a doublet banding pattern in response to TNF-, although overall PKC- levels did not change. Conclusions: TNF--induced growth arrest and differentiation in HPAC cells is associated with the selective downregulation of PKC- and upregulation of PKC-.Presented at the 48th Annual Cancer Symposium of The Society of Surgical Oncology, Boston, Massachusetts, March 23–26, 1995.     

Expression of estrogen receptor-α and -β in anterior vaginal walls of genuine stress incontinent women

Our objective was to study the expression of estrogen receptor (ER) isoforms, ER- and ER-, in the anterior vaginal wall of menopausal and fertile women with genuine stress incontinence (SI) by immunohistochemistry and Western blot analysis. Eighteen menopausal women with SI who either were or were not taking estrogen/progestin replacement therapy and 14 fertile women with SI who either were or were not taking contraceptives were enrolled in the study. Biopsies from the suburethral anterior vaginal wall were obtained at tension-free vaginal tape (TVT) operation. Monoclonal antibody to ER- and polyclonal antibody to ER- were used to stain frozen sections of vaginal tissue. The receptor expressions were scored based on percentage of positive cells. ER- was detected in vaginal epithelial, stromal and smooth muscle cells. In menopausal SI women ER- was detected significantly more frequently in the vaginal walls of estrogen/progestin-treated patients than in those who were untreated. Fertile SI women had significantly higher expression of ER- than menopausal SI women. ER- was not observed in vaginal blood vessels. ER- was detected in epithelial and vascular smooth muscle cells of the vagina. No significant difference in ER- expression was observed between different groups of patients. The expression of ER- was not correlated with that of ER-. Both ER- and - were detected, indicating a potential role for both types of estrogen receptor in the human vaginal wall. The expression of ER-, but not of ER-, in menopausal SI women was regulated by estrogen/progestin replacement therapy. The presence of ER- in vaginal vascular smooth muscle cells raises the possibility of vascular effects of estrogen on the human vaginal wall.Abbreviations ER Estrogen receptor - SI Stress incontinence - TVT Tension-free vaginal tape - HRT Hormone replacement therapy - ABC Avidin–biotin–peroxidase complex - PBS Phosphate buffered saline - DAB DiaminobenzidineEditorial Comment: This was an interesting study in that it addressed the expression of estrogen receptors in incontinent women. There is not a control group of incontinent women. Conclusions regarding mechanisms of action or treatment with estrogen for incontinence cannot be made on the basis of the results. At best this study proves there is expression of estrogen receptors in vaginal tissues, which has been done by prior investigators.     

Plasma disappearance of rabbit α2 HS-glycoprotein and its uptake by bone tissue

Summary Plasma ₂HS-glycoprotein is specifically accumulated in calcified tissues. In the present studies this glycoprotein was isolated from plasma and after iodination with iodine-125 was injected intravenously into young rabbits. The tissue distribution and plasma disappearance rate of this radioactively labeled material were determined. Of the various tissues studied, bone showed the greatest retention of labeled glycoprotein expressed as percentage of the injected dose per gram tissue relative to the plasma content.The rate of loss of iodinated ₂HS-glycoprotein from plasma was similar to that of ₂HS-glycoprotein labeled endogenously by using¹⁴C-glucosamine or³H-glucosamine. The uptake of exogenously labeled³I- ₂HS-glycoprotein into bone tissue expressed as a percentage of the injected dose was similar to that of endogenously labeled¹⁴C- ₂HS-glycoprotein. These results suggest that the¹²⁵I-labeled material can be used to study further the metabolism of ₂HS-glycoprotein by bone tissue.     

The reducing end of αGal oligosaccharides contributes to their efficiency in blocking natural antibodies of human and baboon sera

Synthetic galactosyl oligosaccharides were tested for their ability to inhibit the cytotoxic reaction of human and baboon natural antibodies on PK15 cells in culture. Methyl--Gal gave weak inhibition, Gal1-3Gal substantially inhibited the reaction (400 M), and Gal1-3Gal2-4GlcNAc was ten times more efficient (30 M). The modification from to anomeric configuration of the nonreducing end resulted in a complete loss of activity, while substitutions at the reducing end induced only a partial loss of activity. These observations suggest that natural anti-Gal antibodies recognize the epitope from its nonreducing end, but that substitutions at the reducing terminus can modify the antibody-binding capacity. Modified tri- and tetrasaccharides are better inhibitors than the disaccharide but not as good as Gal1-3Gal1-4GlcNAc. The reducing terminus therefore contributes some energy to the reaction, indicating that certain oligosaccharides will be of more potential clinical use than others.     

Cytokines in human milk: Properties and potential effects upon the mammary gland and the neonate

Epidemiologic and immunologic studies of breastfed and nonbreastfed infants and investigations of certain biologic activities in human milk led to the identification of immunomodulating agents in human milk. Among them were the cytokines interleukin-1 (IL-1); IL-6, IL-8, IL-10, granulocyte-colony stimulating factor, macrophage-colony stimulating factor (M-CSF), tumor necrosis factor-, interferon-, epithelial growth factor (EGF), transforming growth factor- (TGF-), and TGF-2. Inteferon- may originate from T cells in milk; EGF, TGF-, TGF-, M-CSF, IL-6, and IL-8 may be produced by mammary gland epithelium. Based upon their known functions, we hypothesize that cytokines influence the development and immunologic function of the mammary gland and the neonate. Thosein vivo functions remain to be defined by future investigations.