Rapid detection of −α 4.2 deletion of α-thalassemia-2 by polymerase chain reaction

Summary We sequenced part of the X boxes of-thalassemia-1 of Southeast Asia type (- -SEA) with– ^4.2,– ^3.7,– ^G-Taichung, and ^CS. We found the X box of– ^3.7 belonged to the X box of 2 globin gene and the X box of ^cs contained X boxes of both al and2 globin gene, whereas the X box of– ^4.2 and– ^G-Taichung was a hybrid of X boxes of 2 and 1 globin gene. We also found there are two types of– ^4.2 deletion; type 1 is a common type of– ^4.2 deletion and type 2 is linkage to– ^G-Taichung. We used a combination of two methods, the amplification refractory mutation system (ARMS) and the amplified created restriction sites (ACRS), to amplify the hybrids of X boxes specifically. The upstream primer for X box of2 globin gene was designed following the standard ARMS procedure to amplify the X segment of the-globin gene. The downstream primer was designed according to the ACRS method to check the specificity of PCR products. Using this approach, we can diagnose the different types of– ^4.2 deletion. This kind of approach can also be used to amplify the specific region from the cluster of highly homologous genes.     

Lack of α2-Antiplasmin Enhances ADP Induced Platelet Micro-Aggregation through the Presence of Excess Active Plasmin in Mice

Background: The role of 2-antiplasmin (2-AP) on platelet aggregation was investigated using mice deficient in 2-AP (2-AP^–/–) or using wild type mice (2-AP^+/+). Methods: Blood samples were taken from each mouse under anesthesia with ether and platelet rich plasma (PRP) was prepared. Platelet aggregation induced by various doses of ADP (0.3–30 M) was detected using a laser-light scattering (LS) system. Aggregated forms were observed using a scanning electron microscopy (SEM). Results: Dose-dependent platelet aggregation was not different in both types of mice. However, platelet micro-aggregate formation in 2-AP^–/– mice induced by low dose of ADP (1.0 M) markedly increased compared to the situation in wild type mice. Aggregated form detected by SEM showed supported data from LS analysis. When washed platelets of 2-AP^+/+ mice were resuspended in plasma of 2-AP^–/– mice, platelet micro-aggregation was also increased. On the contrary, when washed platelets of 2-AP^–/– mice were suspended in plasma of 2-AP^+/+ mice, platelet micro-aggregation did not change. In separate experiments, tPA (1.0 g/ml) was added to PRP before the stimulation of ADP. tPA had no effect on platelet aggregation in 2-AP^+/+ mice, however platelet micro-aggregation in 2-AP^–/– mice was markedly increased by the treatment with tPA. Moreover, the amount of released ATP from stimulated platelets was increased in 2-AP^–/– mice treated with tPA. Conclusion: Lack of 2-AP increased platelet micro-aggregation, and plasmin plays an important role in the formation of platelet aggregation when 2-AP knockout mice are used. Consequently, the reduction of 2-AP could be a risk factor for the activation of platelets resulting in thrombus formation.     

Hepatic expression of interferon-α in chronic hepatitis B virus infection

Hepatic expression of interferon- (IFN-) was examined by immunohistochemistry in 90 Chinese patients (M/F 67:23, age: 14–69) with a spectrum of hepatitis B virus (HBV)-related chronic liver diseases. Immunoreactive IFN- was detected in sinusoidal cells in 79 patients (88%) and in mononuclear cells in 59 patients (65.6%). Patients with active liver diseases (chronic active hepatitis, active cirrhosis,N=55) had a higher level of IFN- expression compared to patients with inactive histology (N=35; sinusoidal cells,P<0.01; mononuclear cells,P<0.01). Cytoplasmic HBsAg, nuclear HBcAg, and cytoplasmic HBcAg were detected in 79 (88%), 42 (47%), and 23 (27%) patients respectively. Expression of IFN- in mononuclear cells correlated with the expression of cytoplasmic HBcAg (P<0.05) but not with nuclear HBcAg or cytoplasmic HBsAg. When the patients were divided into four different phases according to the natural history of chronic HBV infection, patients in the active liver disease phase had higher IFN- expression compared to the immunotolerant and late phase patients (P<0.01). Using double immunohistochemical staining, both IFN- and cytoplasmic HBcAg were frequently detected near inflammatory infiltrates but no correlation existed between the hepatic expression of HBsAg and IFN-. These data indicate that IFN- is expressed in the liver in HBV-related active liver diseases and that the reported suboptimal production of IFN- by PBMC in HBV-related chronic active liver diseases may be due to a redistribution of the IFN--producing mononuclear cells into the liver, the site of inflammation.     

Interactive influences of peroxisome proliferator-activated receptor α activation and glucocorticoids on pancreatic beta cell compensation in insulin resistance induced by dietary saturated fat in the rat

Aims/hypothesis We sought to elucidate whether excess glucocorticoids and increased dietary lipids act synergistically to impair glucose tolerance and, if so, whether activation of peroxisome proliferator-activated receptor (PPAR) has an adverse or beneficial effect on glucose tolerance.Methods Dexamethasone (100 g kg^–1 body weight day^–1; 5 days) was administered to insulin-resistant rats fed a high-saturated-fat (HF) diet for 4weeks. The PPAR agonist WY14643 was administered (50 mg kg^–1 body weight intraperitoneally) 24 h before sampling. Glucose-stimulated insulin secretion (GSIS) was assessed in vivo after an acute glucose bolus injection, and in vitro using step-up and step-down islet perifusions.Results Although neither PPAR activation nor dexamethasone alone affected fasting glycaemia in the HF group, dexamethasone in combination with PPAR activation elicited marked postabsorptive hyperglycaemia. Dexamethasone treatment of HF rats had little effect on GSIS after an acute glucose challenge in vivo, but induced glucose intolerance. PPAR activation augmented GSIS in dexamethasone-treated HF rats in vivo, restoring glucose tolerance. Contrasting with data obtained in vivo, greatly enhanced peak rates of GSIS were observed ex vivo in perifusions of islets from dexamethasone-treated HF rats compared with those from untreated HF rats, an effect attenuated by antecedent PPAR activation.Conclusions/interpretation The study demonstrates that glucocorticoid excess precipitates the development of glucose intolerance in rats maintained on a high-saturated-fat diet. It does this by interrupting the negative feedback loop between insulin sensitivity and secretion in vivo, such that further enhancement of compensatory insulin secretion is not possible. PPAR activation restores the coupling between insulin secretion and action.     

Increased interleukin 6 production by bronchoalveolar lavage cells in patients with active sarcoidosis

Alveolitis of sarcoidosis is characterized by activated alveolar macrophages (AMs) and T cells. The mediators interleukin-1 (IL-1) and interleukin 6 (IL-6) released by AMs represent essential factors for the progression of the T cells in the cell cycle. The role of IL-1 in pulmonary sarcoidosis has previously been studied; however, the relevance of other mediators (i.e. IL-6) has not yet been evaluated. We measured the spontaneous and lipopolysaccharide (LPS)-induced release of IL-6 and tumor necrosis factor a (TNF) by bronchoalveolar lavage cells (BAL) and peripheral blood mononuclear cells (PBMNC) in 6 control subjects (group A) and in 15 patients with sarcoidosis, 10 with active (group B), 5 with inactive disease (group C). IL-6 as well as TNF were spontaneously released by BAL cells of the active group in significantly greater amounts compared to both other groups; IL-6: A, 165.5 pg/ml/24 hr/10⁶ cells (range, 0–604), B, 946 (0–2467), C, 16.6 (0–83); TNF: A, 162 pg/ml/24 hr/10⁶ cells (0–523), B, 803 (100–17352), C, 100 (0–379). In all groups autologous PBMNC proved to be quiescent, releasing only baseline levels of the cytokines tested. After stimulation with LPS all these cells released great quantities of IL-6 and TNF. In active disease a positive correlation between IL-6 and TNF release was observed (r = 0.77, p < 0.02). The present study documents that in active sarcoidosis the spontaneous release of IL-6 by BAL cells parallels the spontaneous release of TNF. IL-6 is capable of initiating the proliferation and activation of T cells in the lung. Offprint requests to: J. Müller-Quernheim     

α-Adrenergic responses of isolated canine coronary microvessels

Although -adrenergic activation is known to increase coronary microvascular resistance in vivo, the magnitude of its segmental microvascular consequences is not well understood. Quantification of these effects in vivo is hindered by escape mechanisms that minimize the influences of constrictors, and alterations in flow and pressure, which effect microvascular tone by shear stress-dependent and myogenic mechanisms, respectively. To eliminate these confounding influences, we have studied responses in vitro under conditions with these variables controlled. We evaluated the diameter changes of isolated canine coronary arterioles (110±12 m, n=35) and venules (98±7 m, n=9) in response to -adrenergic activation by norepinephrine (10^–10 to 10^–4 M) in the presence of -adrenergic blockade by alprenolol (10^–6 M). In contrast to the situation in vivo, -adrenergic activation did not constrict isolated coronary arterioles, but constricted isolated coronary venules in a dose-dependent manner over a range of 10^–10 to 10^–4 M (–27 ±3% maximum diameter change). Coronary arteriolar -adrenergic constriction was not promoted by 1) subthreshold or vasoactive doses of the vasoconstrictors KCl, angiotensin II, U46619, endothelin-1, neuropeptide Y or arginine vasopressin, 2) inhibition of the presynaptic uptake of norepinephrine by imipramine (10^–6 M), 3) inhibition of EDRF synthesis by N^g-monomethyl-L-arginine (10^–5 M) or 4) inhibition of prostaglandin synthesis by indomethacin (10^–5 M). Furthermore, -adrenergic activation did not modify microvascular dilatation by adenosine (10^–9 to 10^–4 M) or nitroglycerin (10^–9 to 10^–4 M), suggesting that -adrenergic constriction in vivo is not due to attenuation of cAMP or cGMP-dependent mechanisms of coronary dilatation. In contrast to the lack of constriction in coronary arterioles, canine skeletal muscle arterioles exhibited significant -adrenergic constriction (–80±4%), maximum diameter change). The coronary venular -adrenergic constriction was significantly inhibited by both the ₁-and ₂-adrenergic receptor antagonists, prazosin (10^–8 M) and rauwolscine (10^–7 M), indicating a mixed population of ₁-and ₂-adrenergic receptors. These results suggest that coronary arterioles, but not venules, lose -adrenergic responsiveness during isolation and cannulation, or that the primary coronary microvascular response to -adrenergic activation is venular constriction.     

Correlation Between Stellate Cell Activation and Serum Fibrosis Markers in Choline-Deficient l-Amino Acid-Defined Diet-Induced Rat Liver Fibrosis

The aim of this study was to investigate the association of stellate cell activation with serum fibrosis markers in a rat model of hepatic fibrosis prepared using a choline-deficient l-amino acid (CDAA) defined diet. CDAA diet administration resulted in increased liver hydroxyproline contents in a time-dependent manner with activated stellate cells, expressing -smooth muscle actin (-SMA) as well as increased serum concentrations of amino-terminal procollagen type III peptide (PIIIP) and the 7S fragment of type IV collagen. Hydroxyproline content of the liver showed a closer correlation with the serum 7S (r = 0.75, P < 0.01) concentration than with the serum PIIIP (r = 0.51, P < 0.01) concentration. The percent area of -SMA-positive cells showed stronger correlation with the serum PIIIP concentration (r = 0.85, P < 0.01) than with the 7S concentration (r = 0.50, P < 0.01). These results indicate that the serum PIIIP concentration reflects the activity of fibrogenesis, while the serum 7S concentration reflects the accumulation of collagen fibers in the liver.     

Autocrine growth stimulation of SW403 colon carcinoma cell line is caused by transforming-growth-factor-α-mediated epidermal growth factor receptor activation

Results of recent studies indicate that some cultured human carcinoma cell lines are capable of proliferating autonomously in serum-free medium as a result of the synthesis and secretion of transforming growth factor (TGF). TGF interacts with epidermal growth factor receptor (EGFR) and induces its activation. In an attempt to extend these observations, we evaluated TGF-mediated autonomous growth and constitutive EGFR activation in the human adenocarcinoma cell line SW403. The cell line shows synthesis of EGF receptors and TGF but not EGF, and exhibits constitutive phosphorylation of the 170-kDa EGFR. Use of blocking anti-EGFR monoclonal antibodies (mAb) inhibits autonomous growth of SW403 cells and leads to a significant reduction of receptor phosphorylation. The inhibitory effect of the blocking anti-EGFR mAb is reversible upon addition of TGF. In contrast, autonomous proliferation of SW403 cells is not inhibited by addition of neutralizing anti-EGF mAb. Our findings suggest that the proliferation of cells of the human SW403 adenocarcinoma cell line is regulated by an autocrine TGF loop and that this regulatory pathway can be interrupted by using anti-EGFR mAb.Abbreviations EGF epidermal growth factor - EGFR epidermal growth factor receptor - mAb monoclonal antibody - TGF transforming growth factor      

β-Adrenoceptors, but not α-adrenoceptors, stimulate AMP-activated protein kinase in brown adipocytes independently of uncoupling protein-1

Aims/hypothesis Brown adipocytes provide a potentially important model system for understanding AMP-activated protein kinase (AMPK) regulation, where adrenergic stimulation leads to mitochondrial uncoupling through uncoupling protein-1 (UCP1) activity. AMPK is a sensor of energy homeostasis and has been implicated in glucose and lipid metabolism in several insulin-sensitive tissues. The aim of this study was to characterise the potential role of AMPK in adrenergically mediated glucose uptake and to find out whether UCP1 is involved in the adrenergic activation of AMPK.Methods We used primary brown adipocytes differentiated in culture and measured AMPK phosphorylation and glucose uptake following adrenergic activation.Results Treatment of adipocytes with noradrenaline (norepinephrine) caused phosphorylation of AMPK via -adrenoceptors and not ₁- or ₂-adrenoceptors. This effect was not ₃-adrenoceptor specific, since responses remained intact in adipocytes from ₃-adrenoceptor knock-out mice. These effects were also mimicked by forskolin and cAMP analogues. Treatment of cells with adenine 8--d-arabinofuranoside, an AMPK inhibitor, partially blocked -adrenoceptor-mediated increases in glucose uptake. Brown adipocytes are characterised by the production of UCP1, which can uncouple the mitochondria. Using adipocytes from Ucp1^+/+ and Ucp1^–/– mice, we showed that noradrenaline-mediated phosphorylation of AMPK does not require the presence or activity of UCP1.Conclusions/interpretation These results suggest a pathway where increases in cAMP mediated by -adrenoceptors leads to activation of AMPK in brown adipocytes, which contributes in part to -adrenoceptor-mediated increases in glucose uptake, an effect independent of the presence or function of UCP1.     

Lack of association of tumor necrosis factor-alpha gene polymorphisms with disease susceptibility and severity in Behçet’s disease

Although it has been reported that the MHC class I molecule, HLA-B51, is a risk factor for Behçets disease (BD), contribution of the tumor necrosis factor (TNF) genes, which are located in the vicinity of the HLA-B locus, to the genetic susceptibility for BD has yet to be elucidated. The purpose of this study was to analyze the effect of TNF- promoter polymorphisms at positions –308, –238 and –376 on the susceptibility, severity and clinical features of BD. The TNF- gene sequences from 107 patients with BD and 102 healthy subjects were amplified by the polymerase chain reaction. Sequence analysis of the TNF- gene locus, which contains promoter polymorphisms at positions –376, –308, and –238, was performed with a DNA sequencing kit on automated sequencer. The patients were classified according to disease severity and clinical features. Serum TNF- level in the study groups was measured by sandwich enzyme immunoassay. In patients with BD the frequencies of TNF- –308 (19.4% vs 18.4%), –238 (3.7% vs 5.9%), and –376 (0.9% vs 2.9%) gene polymorphisms were not found to be significantly different from those in healthy subjects. The TNF- gene polymorphisms did not show any association with disease severity or clinical features. Serum TNF- level was significantly higher in patients with BD than in healthy controls (3.10±1.45 pg/ml vs 2.43±1.94 pg/ml, P < 0.01). Serum TNF- level was not found to be significantly associated with disease severity, activity, clinical findings and TNF- genotypes. The results of this study suggest that the TNF- gene polymorphisms are unlikely to play an important role in the pathogenesis and severity of BD.     

Interleukin-2-activated killer cell activity in colorectal tumor patients: evaluation of in vitro effects by prothymosinα1

The effects of prothymosin 1 (Pro 1) in combination with interleukin-2 (IL-2) on peripheral blood lymphocytes from 50 colorectal tumor patients at different stages were studied with respect to immunocytotoxicity, adhesion to cultured SW620 colon carcinoma cells, secretion of cytokines and expression of adhesion and surface marker molecules. On average, the patients showed lower natural killer (NK) cell activity than healthy donors, which was associated with a lower adhesion capacity to the tumor target cells. The NK cell activity of the patients was inversely related to the tumor stage. The generation of lymphokine(IL-2)-activated killer (LAK) cell activity was found to be comparable on lymphocytes from healthy individuals and patients and was not correlated to tumor stage. Pro 1 stimulated patients' LAK cell activity only, primarily at the early stage (Dukes A/B). The Pro 1 effect was associated with an increased adhesion of lymphocytes to tumor target cells and an increased secretion of the deficient IL-2-induced IFN secretion. No significant effects on the low level of TNF secretion was noted. By flow cytometry, Pro 1 in combination with IL-2 augmented the expression of the NK cell markers CD56, CD16/56, the subset CD3/16/56 and CD25 on lymphocytes of the patients. In contrast, Pro 1 was equally effective by increasing the expression of CD18 and CD11a, on lymphocytes from the patients and from normal controls. In conclusion, Pro 1, in combination with IL-2, can partially normalize lymphocyte deficiencies of colon cancer patients in vitro. This potential might provide an experimental basis for applying Pro 1 or related thymic peptides in selected immunotherapies against colorectal tumors.Abbreviations Pro 1 prothymosin 1 - NK natural killer - LAK lymphokine-activated killer - IL-2 interleukin-2 - TNF tumor necrosis factor - IFN interferon - PBL peripheral blood lymphocytes - ELISA enzyme-linked immunosorbent assay     

Triplication (/ααα<Superscript>Anti3.7</Superscript>) or deletion (-α<Superscript>3.7</Superscript>/) association in Argentinian β-thalassemic carriers

Prevalence of alpha gene triplication or deletion in -thalassemia carriers was studied in 109 unrelated individuals in Rosario, Argentina. In different populations -^3.7 allele presents a higher prevalence than ^anti3.7; thus, -thalassemia associated with -thalassemia is more frequently observed. Nevertheless, this event was detected in only one case (0.9%), while the association with alpha triplication was present in two subjects (1.8%).     

α-Smooth-muscle actin expression in normal and fibrotic human livers

To determine the significance of the expression of -smooth-muscle actin in the fibrotic human liver, normal and diseased livers were stained with anti--smooth-muscle-actin antibody by an immunoperoxidase method. Vitamin A-containing lipocytes were also identified by the modified Kupffer's gold chloride method. In the normal human liver, lipocytes as well as vascular smooth muscle cells expressed -smooth-muscle actin. In alcoholic liver disease, there was an increase in the cells positive for -smooth-muscle actin adjacent to the fibrotic areas, but the response of lipocytes to the gold chloride reaction diminished. In chronic hepatitis, the cells positive for -smooth-muscle actin increased around the enlarged portal areas, and the response to the gold chloride reaction did not change appreciably. An increase in the cells positive for -smooth-muscle actin was associated with the progression of hepatic fibrosis in the liver of patients with alcoholic liver disease and chronic hepatitis.     

Effects of human α-interferon on granulocyte-macrophage progenitor cells (CFU-GM) in vitro

Summary Two preparations of human interferon (IFN)- were assessed for their influence on granulocyte-macrophage progenitor cells (CFU-GM) in vitro. Both highly purified human IFN- Ly and recombinant IFN- 2a suppressed CFU-GM colony formation in a dose-dependent manner using low-density bone-marrow target cells. Suppression of CFU-GM colony formation was accompanied by an increase in clusters. However, depletion of monocytes, T lymphocytes and B lymphocytes from low-density bone-marrow cells resulted in insensitivity of progenitor cells to IFN-. These results demonstrate that the effects of human IFN- on myeloid progenitor cells (CFU-GM) are mediated by accessory cells within the bone marrow.     

Crohn’s disease complicated by adult-onset Still’s disease

A 31-year-old man with Crohns disease developed arthritis, spiking fever, and skin rash indistinguishable from that of adult-onset Stills disease. He was admitted to our hospital because of a periumbilical intestinal skin fistula. Crohns disease had been diagnosed in 1991, and had required intestinal resection twice, and schizophrenia had been diagnosed in 1993. He developed polyarthritis and spiking fever, accompanied by a macular skin rash on both forearms. Marked hepatosplenomegaly and bilateral pleural effusion were detected on computed tomography examination. These findings are indistinguishable from those of adult-onset Stills disease. Because his mental status had deteriorated following high-dose prednisolone on a previous admission, he was treated with an immunosuppressive agent on this occasion, with the treatment being successful. This is the first report of adult-onset Stills disease complicating Crohns disease. In patients with Crohns disease, polyarthritis and skin rash can easily be misdiagnosed as enteropathic arthritis with erythema nodosum associated with the Crohns disease. Although adult-onset Stills disease may not be fatal, early diagnosis is important because it can, in rare cases, result in life-threatening complications.     

Short-term inhibition of peroxisome proliferator-activated receptor-γ coactivator-1α expression reverses diet-induced diabetes mellitus and hepatic steatosis in mice

Aims/hypothesis The coactivator of nuclear receptors, peroxisome proliferator-activated receptor- coactivator-1 (PGC-1) has been implicated in a series of events that contribute to the control of glucose metabolism. We have recently reported the use of a PGC-1 antisense oligonucleotide (PGC-1AS) that inhibits up to 60% of PGC-1 expression in pancreatic islets, leading to increased insulin secretion. This oligonucleotide was used in this study to try to ameliorate diet-induced type 2 diabetes in a genetically predisposed mouse strain (Swiss mice).Materials and methods Glucose and insulin tolerance tests, euglycaemic–hyperinsulinaemic clamp, immunoprecipitation assays, immunoblotting assays and immunohistochemistry were used in this investigation.Results Swiss mice became obese and overtly diabetic after 8 weeks of feeding with chow containing 24% saturated fat. One daily dose (1.0 nmol) of PGC-1AS significantly reduced glucose and increased insulin blood levels without affecting food intake and body weight. These effects were accompanied by a reduced area under the glucose curve during an intraperitoneal glucose tolerance test, an increased constant of glucose decay (K_itt) during an insulin tolerance test, and an increased glucose consumption rate during a euglycaemic–hyperinsulinaemic clamp. Moreover, mice treated with PGC-1AS presented an outstanding reduction of macroscopic and microscopic features of hepatic steatosis. These effects were accompanied by reduced expression or function of a series of proteins involved in lipogenesis.Conclusions/interpretation PGC-1 is an attractive target for pharmacological therapeutics in type 2 diabetes mellitus and diet-induced hepatic steatosis.     

Liver microsomal Δ6 and Δ5 linoleic acid desaturation in female BB rats

Summary Rat liver microsomal 6 and 5 desaturation are defective in experimental diabetes, but this defect is correctable with insulin treatment. Rat liver fatty acid composition and 6 and 5 desaturation were studied in the spontaneously diabetic adult female Bio-Breeding (BB) rat. Control Wistar rats and BB rats (4 weeks of diabetes), that received insulin (1 IU·100 g body weight^–1·day^–1), were killed 20 h after the last insulin injection. 6 and 5 desaturase activities were estimated from the incubation of liver microsomes with (1-¹⁴C) 18:2, n–6 or (2-¹⁴C) 20:3, n–6, respectively, and the fatty acid composition of the liver and microsomal liver lipids were investigated. Under experimental conditions 6 and 5 desaturase activities were unchanged in the BB rats when compared to the control rats. Impairment of the liver fatty acid composition of diabetic BB rats is not consistent with normal desaturase activity and may be explained by factors other than desaturation disturbance.     

Transforming Growth Factor-α Precursors in Human Colon Carcinoma Cells

Among the proteins of the epidermal growth factor family, transforming growth factor- (TGF-) may be an especially reliable indicator of metastasis or prognosis in human colorectal carcinomas. Moreover, anomalous forms of TGF- have been detected in several tissues of cancer origin, suggesting a role of these forms in the development of the disease. This study was designed to identify the presence of TGF- precursors in different colon cancer cell lines by mean of immunocytochemistry and western blotting techniques. Pro-TGF- was detected in all cell lines tested. Staining for pro-TGF- was observed in cytoplasm. Monoclonal antibody to TGF- detected two bands of 20 and 21 kDa. Polyclonal antibody to pro-TGF- revealed five bands ranging from 15 to 24 kDa. All these proteins were also detected in nonmalignant cells expressing a transfected rat pro-TGF- gene. In conclusions, transformation in these human colon carcinoma cells is not due to the presence of anomalous forms of TGF- precursors.     

Presence of interferon and anti-interferon in patients with systemic lupus erythematosus

Summary Sera from 61 patients with systemic lupus erythematosus (SLE) were serially screened over a period of at least 2 years for IFN and anti-IFN antibodies. IFN concentrations were measured both with a cytopathic effect assay and a more sensitive radioimmunoassay. Of the patients 15% (9/61) had IFN in their serum at one or more occasions as measured in the bioassay (6 IU/ml); employing a RIA (1 IU/ml) 28% (17/61) of the patients studied were positive for IFN-. Fifteen patients had a measurable interferonemia over 2–16 months; only two patients had detectable IFN in their serum at only one occasion. In five patients, hourly and daily variations of the IFN titer as measured by RIA were found to amount to less than 80%. The IFN activity found in these sera was characterized as IFN- by means of acid stability, cross-reactivity on heterologous cells, trypsin sensitivity, and neutralization by homologous and heterologous antisera. IFN antibodies were quantified with a neutralization bioassay, an ELISA, and a radioimmunoassay. Of the 61 patients 5% (3) possessed high titers of anti-IFN antibodies which persisted over 2 years. The IFN- antibody positive patients had an inactive form of the disease over years without visceral involvement but decreased serum complement levels (C4, C3, CH50) and repeated episodes of Quincke-like edema.     

Cytotoxic effect of a fusion protein from transforming growth factora andPseudomonas exotoxin on rat and human bladder carcinoma cells in vitro

A protein formed by fusion of transforming growth factor withPseudomonas exotoxin (TGF-PE40) has been shown to have the ability to kill or inhibit the growth of several carcinoma cell lines. This study was designed to evaluate thein vitro cytotoxic effects of TGF-PE40 on rat and human bladder carcinoma cell lines with different biological potential, and normal rat urothelial cells. The rat cell lines used were D44c, LMC19, and MYU3L, which were established in our laboratory. Human cell lines used were RT4, T24, and 253J. As a normal control, we used the first-passage culture of normal rat bladder urothelium (RU-P1). We examined the number and affinity of epidermal growth factor receptors (EGFR) in these cells, the ability of TGF-PE40 to bind EGFR, and the cytotoxic effect of TGF-PE40 and PE40. Rat cell lines, D44c, LMC19, and MYU3L (EGFR=4.9×10³–11.4×10³/cell) had ED₅₀ values (the concentration of TGF-PE40 needed to reduce the viable cell population by 50%) of 180 pM, 540 pM and 6000 pM respectively; forc ₁ (the concentration required to achieve complete inhibition of growth under continuous serum stimulation) TGF-PE40 concentrations of 10⁴ pM, 10⁴ pM and higher than 10⁴ pM respectively were required. Human cell lines, RT4, T24, and 253J (EGFR=32×10³–126×10³/cell) had ED₅₀ values of 20 pM, 66 pM, and 330 pM respectively and T24 showedc ₁ values of 10³ pM. RU-P1 (EGFR =92.6×10³/cell) had the highest ED₅₀ value of 8000 pM. These data indicate that the susceptibility to TGF-PE40 does not always depend on the number of EGFR, that cells having a relatively small number of EGFR respond well to TGF-PE40, and that normal urothelial cells are more resistant to TGF-PE40 than are cancer cells. The differential effect of TGF-PE40 on normal and neoplastic cells provides a rational basis for its use in vivo to control tumor growth.Supported by National Institutes of Health Grant CA14649