Prospective evaluation of interferon-α in treatment of chronic active Crohn's disease

Several case reports suggested good effects of interferon- in patients with Crohn's disease. In addition, a decreased production of interferon- in Crohn's disease has been shownin vitro. Treatment with interferon- may activate intestinal natural killer cells and down-regulate the overproduction of inflammatory cytokines like interleukin-6 in Crohn's disease. To evaluate the clinical efficacy of interferon-, we treated 12 patients with a chronic active course of Crohn's disease with recombinant human interferon- prospectively for 24 weeks. Prednisolone was continuously tapered and discontinued at week 12. The end point of the study was the prevention of worsening of clinical symptoms defined with the Crohn's disease activity index and was monitored by acute-phase proteins, interleukin-6 serum concentrations, and endoscopy. The biochemical activity of interferon- was measured by 2,5-oligo adenylate serum levels. The end point of the study was reached in four patients (33%). In these patients the final Crohn's disease activity index was above 150, which means that they did not achieve clinical remission. All other patients (66%) did not respond to interferon- and had to be withdrawn prematurely. Interferon- did not show any beneficial effect on interleukin-6 or acute-phase protein concentrations and on endoscopic activity. The 2,5-oligo adenylate levels continuously increased during interferon therapy. Considerable side effects were noted. These results fail to demonstrate a therapeutic role of interferon- in chronic active Crohn's disease.     

Rapid molecular characterization of Hb H disease in Chinese by polymerase chain reaction

Summary We have developed a rapid method to molecularly distinguish different types of Hb H disease. The study depended on (a) most of the Hb H disease in Taiwan having an-thalassemia-1 of the Southeast Asia type (-SEA) in one allele and (b) the differences of X box of-globin gene cluster in the other allele. To detect the -SEA allele, we utilized the primers located on either side of the breakpoint to do PCR, then characterized the amplified products. For the other allele, we sequenced part of the X box, and found that bases –2803 to –2461 of the X box of – ^3.7 belonged to the X box of ₂ globin gene. In – ^4.2, the bases belonged to the X box of ₁ globin gene, whereas in ^cs it contained both X boxes of ₁ and ₂ globin genes. There was anMboII site at this region of the X box of ₂ globin gene. We utilized PCR to amplify this region and digested it with restriction enzymeMboII, then combined it with another PCR of different primer pairs to molecularly diagnose different types of Hb H disease. One hundred and one cases of Hb H disease from different families were studied: all of the cases had one allele of -SEA deletion, while the other allele showed that 52/101 were – ^3.7, 41/101 were ^cs , 7/101 were – ^4.2, and 1/101 was – ^G.Taichung. Of 52 cases of Hb H with – ^3.7, 47 were type-I deletion and five were type-II deletion.     

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.     

Regulation of the release of tumour necrosis factor (TNF)α and soluble TNF Receptor by γ irradiation and interferon γ in Ewing's sarcoma/peripheral primitive neuroectodermal tumour cells

This study analyses the production of tumour necrosis factor (TNF) and soluble TNF receptor (sTNF-R) before and after exposure to irradiation and interferon (IFN) in 12 cell lines derived from Ewing's sarcoma (ES)/peripheral primitive neuroectodermal tumours (pPNET). Supernatants from ES/pPNET cell cultures were tested in a TNF-specific amplified enzyme-linked immunosorbent assay (ELISA), a bioassay, and sTNF-R_p55 and sTNF-R_p75 ELISA. The tumour cell lines released minimal amounts of TNF, prominent amounts of sTNF-R_p55 (7/12 cell lines) and no sTNF-R_p75. Exposure to irradiation (5 Gy) either induced (3/12) cell lines) or up-regulated (3/12 cell lines) TNF release without changing sTNF-R_p55 and sTNF-R_p75 levels. Priming of cultures with recombinant human IFN (rhIFN) markedly enhanced TNF secretion in the radiation-responsive cell lines and had no influence on sTNF-R_p55 and sTNF-R_p75 levels. rhIFN affected the magnitude rather than the sensitivity of the radiation response. The TNF secreted was bioactive, as shown by its cytotoxic effect of WEHI-164 cells, and neutralization of its activity by anti-TNF monoclonal antibody. Herbimycin A (a tyrosine-specific protein kinase inhibitor) but not calphostin C (a protein kinase C inhibitor), H89 (a protein kinase A inhibitor), AACOCF3 (a specific inhibitor of phospholipase A₂) and MK-886 (a specific inhibitor of 5-lipoxygenase) abrogated -irradiation-stimulated TNF release. The antioxidantsN-acetylcysteine, nordihydroguaiaretic acid and mepacrine dose-dependently inhibited -irradiation-mediated TNF production. Collectively our findings indicate that IFN priming potentiates the secretion of bioactive TNF by ES/pPNET cells in response to irradiation without affecting sTNF-R release. The data suggest a requirement for protein tyrosine kinase activity and a role for reactive oxygen species in the -irradiation-mediated intracellular signalling pathway leading to TNF production.     

A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease type D (MPS IIID)

Summary 4-Methylumbelliferyl--N-acetylglucosamine 6-sulphate was synthesized and shown to be a substrate for the lysosomalN-acetylglucosamine-6-sulphate sulphatase (GlcNAc-6S sulphatase). Fibroblasts and leukocytes from 3 different Sanfilippo D patients showed <1% of mean normal GlcNAc-6S sulphatase activity. The enzymatic liberation of the fluorochrome from 4-methyl-umbelliferyl--N-acetylglucosamine 6-sulphate requires the sequential action of the GlcNAc-6S sulphatase and -N-acetylglucosaminidase. A normal level of -N-acetylglucosaminidase activity was insufficient to complete the hydrolysis of the reaction intermediate 4-methylumbelliferyl--N-acetylgluco-saminide formed by the GlcNAc-6S sulphatase. A second incubation in the presence of excess -N-acetyglucosaminidase is needed to avoid underestimation of the GlcNAc-6S sulphatase activity.     

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      

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     

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.     

New tumor necrosis factor-α-inducing protein released from<Emphasis Type="Italic"> Helicobacter pylori</Emphasis> for gastric cancer progression

Purpose To investigate the association between Helicobacter pylori infection and its inflammatory reaction in gastritis, gastric ulcer, and gastric cancer, a new tumor necrosis factor- (TNF-)-inducing protein of H. pylori was studied.Methods The HP0596 gene of H. pylori was identified as the TNF--inducing protein (Tip) gene from genome sequence of H. pylori strain 26695. Using recombinant Tip (rTip) and deleted Tip (rdel-Tip) proteins, the latter of which lacks six amino acids containing two cysteines in the N-terminal domain, we examined their activities in TNF- gene expression and NF-B activation in both Bhas 42 (v-H-ras transfected BALB/3T3) cells and mouse gastric epithelial cell line MGT-40, and in vitro transformation of Bhas 42 cells.Results Tip protein as a homodimer form (38 kDa) was found in both extracts and culture medium of various H. pylori strains. rTip significantly induced TNF- gene expression and NF-B activation in both Bhas 42 cells and MGT-40, and induced in vitro transformation of Bhas 42 cells. However, rdel-Tip did not. Treatment with MG-132, a proteasome inhibitor, inhibited translocation of NF-B p65, and abrogated TNF- induction induced by Tip protein.Conclusion Tip is a new carcinogenic factor released from H. pylori mediated through NF-B activation.     

Enzymatic determination of serum 3α-sulfated bile acids concentration with bile acid 3α-sulfate sulfohydrolase

A newly developed enzymatic method for determining urinary 3-sulfated bile acids was used to measure serum 3-sulfated bile acid levels in 114 patients with hepatobiliary diseases and 56 healthy subjects. The lowest measurable amount of the 3-sulfated bile acids was 0.5 µmol/liter. The standard curves for glycolithocholic acid 3-sulfate, glycoursodeoxycholic acid 3-sulfate, and lithocholic acid 3-sulfate were linear from 0.5 to 250 µmol/liter. Specificity of the assay was satisfactory and intra- and interassay variations ranged from 0.8 to 4.4% and from 1.2 to 7.9%, respectively. Analytical recovery was more than 91%. The values obtained by this assay were well correlated with those by gas-liquid chromatography measurement (r=0.91,P<0.01). The fasting serum 3-sulfated bile acids level in healthy subjects ranged from undetectable to 1.9 µmol/liter (mean±se; 0.9±0.1 µmol/liter). The percentage of 3-sulfated bile acids in total bile acids (sum of 3-sulfated and 3-hydroxy bile acids) in serum was 16.8±1.5%. In subjects with hepatobiliary diseases, serum 3-sulfated bile acids levels were elevated; however, the percentage of 3-sulfated bile acids in total bile acids was decreased and correlated with the severity of hepatocellular insufficiency. This enzymatic assay is simple, rapid, and accurate for the determination of serum 3-sulfated bile acids.     

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.     

Changes in expression levels of genes involved in fatty acid metabolism: upregulation of all three members of the PPAR family (α, γ, δ) and the newly described adiponectin receptor 2, but not adiponectin receptor 1 during neonatal cardiac development of the rat

During neonatal cardiac development, the heart changes its substrate preference from glucose to fatty acids. The aim of this study was to investigate the changes in mRNA expression levels of genes involved in the control of cardiac fatty acid metabolism in the transition from neonatal to adult life. Methods mRNA expression levels for peroxisome proliferator activated receptor (PPAR) , and , PPAR co–factor 1 and (PGC–1 and ), 9–cis retinoc–acid–activated receptor , and (RXR , , ), 5–AMP activated protein kinase (AMPK) 1 and 2, adiponectin receptor 1 and 2 (AR 1 and AR 2) were measured in heart tissue of neonatal 0–day, 7–day and 21– day old rats. Results mRNA expression of all three members of the PPAR family were upregulated significantly from day 0 to day 21 ( +117%, +133%, +203%). In addition, m–RNA expression of all RXR isoforms increased from day 0 to day 7 ( +125%, +69%; +41%). AR 2 exhibited a small but significant increase in mRNA expression (+ 46%). Conclusions We were able to demonstrate for the first time that in addition to PPAR, also PPAR and , as well as all RXR isoforms and AR 2 are upregulated in the heart during neonatal development.Drs. Steinmetz and Quentin contributed equally to this publication     

Characterization of integrin subunits,cellular adhesion and tumorgenicity of four human prostate cell lines

Cellular adhesion to extracellular matrix proteins via integrin molecules is a major factor in the process of invasion and metastasis of human tumor cells. Four human prostate cell lines were characterized according to the presence and quantity of integrin subunits, the ability of the cells to attach to extracellular substrates and the capacity of the cells to form tumors in severe combined immunodeficient (SCID) mice. All four human prostate cell lines expressed three to five integrins on their cell surfaces. The DU145, PC3 and 431P cells expressed primarily ₃, ₅, and ₆ integrin at similar levels. These cell lines expressed the subunits ₁, ₃ and ₄ with ₁ predominant. The DU145 cells preferred attachment to fibronectin, followed by laminin and vitronectin. Approximately 50%–60% of the binding of DU145 cells to fibronectin and laminin was dependent on the function of ₅₁ and ₆ respectively. The cell line LNCaP differed in its low expression of the ₃ subunit, 95% of cellular adhesion to fobronectin and laminin being integrin-dependent and its inability to attach to vitronectin, in spite of surface expression of _v3. All the cell lines except for LNCaP readily formed tumors within SCID mice and the expression of ₃, ₆, ₁, and ₄ integrin subunits was preserved in the resulting tumor tissue. The altered adhesion properties of the LNCaP cells may explain their altered tumorigenicity.Abbreviations SCID severe combined immunodeficiency - FITC fluorescein isothiocynate - FACS fluorescence-activated cell sorting - PBS phosphate-buffered saline - FBS fetal bovine serum This work was supported in part by a grant from the Friends of the Arizona Cancer Center, Phoenix Chapter, is ACS grant PDT-388 and CH-467 and CA 56666     

Interferonγ and tumour necrosis factorα induce a synergistic antiproliferative response in human Ewing's sarcoma cells in vitro

Three human cell lines derived from Ewing's sarcoma (RM-82, VH-64, and WE-68) were investigated to establish the influence of recombinant human interferon (rhIFN) and tumour necrosis factor (rhTNF) on cell proliferation and survival and to characterize IFN and TNF receptor expression. Incorporation of [³H]thymidine into cells was inhibited by rhIFN after 24 h of incubation. Half-maximal inhibition was observed with 10–80 U/ml rhIFN. A maximal effect (50%–70% inhibition of cell proliferation) was achieved by treatment of cells with 250 U/ml rhIFN. The influence of rhTNF on proliferation was found to differ among cell lines and varied with the concentration and the duration of exposure of cells to this cytokine. In WE-68 and VH-64 cells [³H]thymidine incorporation was not affected by rhTNF up to 2000 U/ml after 96 h of incubation, where-as in RM-82 cells the incorporation was inhibited by 35% after 48 h of incubation with 100 U/ml rhTNF. However, all cell lines showed a synergistic antiproliferative response to the combination of rhIFN and rhTNF after 24 h of incubation. The human recombinant cytokines interleukin(IL)-1, IL-1, IL-2, IL-3, IL-4, IL-6 and granulocyte/macrophagecolony-stimulating factor, tested alone and in combination with rhIFN and rhTNF, had no influence on cell proliferation. Binding studies in the cell lines with¹²⁵I-rhIFN revealed a dissociation constant (K _d ) of 160–306 pM and approximately 8000–13500 receptors/cell. Binding experiments with¹²⁵I-rhTNF indicated 430–1250 receptors/cell withK _d ranging from 13 pM to 162 pM. These data indicate that, among various cytokines, only IFN and TNF are capable of potently reducing Ewing's sarcoma cell growth in vitro. Our data suggest that IFN alone or in combination with TNF may be useful in the design of novel strategies in Ewing's sarcoma therapy.     

Low Endogenous Prostaglandin E2 Predisposes to Relapsing Inflammation in Experimental Rat Enterocolitis

Intramural injection ofpeptidoglycan-polysaccharide (PG-PS) induces acuteenterocolitis that spontaneously relapses in Lewis butnot Fischer rats. Interleukin-1 (IL-1) and tumornecrosisfactor- (TNF-) induce prostaglandin E₂(PGE₂) secretion, which inhibits secretion ofthese cytokines by macrophages, suggesting an inhibitoryfeedback mechanism. We postulate that Lewis ratsusceptibility to relapse is due to an imbalance betweenprotective prostaglandins and cytokines. Female Fischerand Lewis rats were injected with PG-PS (37.5 g/g)or human serum albumin intramurally. Tissue IL-1 and PGE₂ immunoreactivities andmyeloperoxidase (MPO) activity were determined.Relapsing rats had lower PGE₂ andPGE₂:IL-1 ratios than nonrelapsingrats (P < 0.05). In Fischer rats, 2 mg/kg/day indomethacinpotentiated cecal MPO and IL-1 concentrationsabove PG-PS alone (P < 0.05). Misoprostol treatmentblocked PG-PS-induced IL-1 and MPO and inhibited the potentiating effect of indomethacin on MPOand IL-1 (P < 0.05). In conclusion, increasedendogenous PG may be protective against relapsinginflammation in PG-PS induced enterocolitis, at least partially via inhibition of proinflammatorycytokines. An imbalance between protectiveprostaglandins and proinflammatory cytokines may beinvolved in the pathogenesis of chronic relapsinginflammation in genetically susceptible hosts.     

A fluorimetric enzyme assay for the diagnosis of MPS II (Hunter disease)

4-Methylumbelliferyl--iduronate 2-sulphate was synthesized and shown to be a specific substrate for the lysosomal iduronate-2-sulphate sulphatase (IDS). Fibroblasts (n = 17), leukocytes (n = 3) and plasmas (n = 9) from different MPS II patients showed <5% of mean normal IDS activity. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl--iduronate 2-sulphate requires the sequential action of IDS and -iduronidase. A normal level of -iduronidase activity was insufficient to complete the hydrolysis of the reaction intermediate 4-methylumbelliferyl--iduronide formed by IDS. A second incubation step in the presence of excess purified -iduronidase is needed to avoid underestimation of the IDS activity.     

Ataxia associated with increased plasma concentrations of pristanic acid,phytanic acid and C27 bile acids but normal fibroblast branched-chain fatty acid oxidation

Summary Investigations of peroxisomal function were undertaken in an 8-year-old girl who developed motor difficulties at the age of 3.5 years and went on to develop a progressive ataxia and dysarthria. There were no other neurological abnormalities and she was of normal intelligence. Analysis of plasma very long-chain fatty acids revealed a normal C₂₆ concentration and normal C₂₄/C₂₂ and C₂₆/C₂₂ ratios. Analysis of branched-chain fatty acids showed an elevated plasma phytanic acid concentration of 60 µmol/L (normal<15) and a considerably elevated pristanic acid concentration of 50 µmol/L (normal<2). Plasma concentrations of the C₂₇ bile acids 3,7-dihydroxycholestanoic acid (DHCA) and 3,7,12-trihydroxycholestanoic acid (THCA) and of the C₂₉-dicarboxylic acid were also increased. We postulated that these results might be due to deficiency of the peroxisomal branched-chain acyl-CoA oxidase, but when oxidation of branched-chain fatty acids was studied in cultured skin fibroblasts it was found to be normal. Alternative explanations for the accumulation of branched-chain substrates for peroxisomal-oxidation are discussed. Treatment with a low-phytanic acid diet arrested the progression of the ataxia and led to a slight improvement.     

Serum cytokines in patients with rheumatoid arthritis

Serum cytokines such as interleukin 1 (IL-1), interferon (IFN-), and tumor necrosis factor (TNF) were measured in 40 patients with rheumatoid arthritis (RA). In the 40 patients studied, serum IL-1 was detected in 5 patients, IFN- in 10 patients, and TNF in 20 patients. The IL-1-positive group showed increased values of activity indices compared to the IL-1-negative group. Values of serum IFN- correlated well with the number of peripheral blood lymphocytes and CD3⁺ cells and with the percentage of CD3⁺ CD26⁺ cells. Values of serum TNF correlated positively with the number of peripheral blood monocytes and the percentage of CD3⁺ HLA-DR⁺ and CD3⁺ CD25⁺ cells. These results indicated that serum IL-1 in RA patients reflects the activity of RA, while the serum IFN- and TNF in RA patients may be related to circulating activated lymphocytes and monocytes, respectively.     

Overproduction of inhibitory hematopoietic cytokines by lipopolysaccharide-activated peripheral blood mononuclear cells in patients with aplastic anemia

The aim of this study was to measure the level of cytokines produced by peripheral blood mononuclear cells (PBMNC) in patients with aplastic anemia (AA) and to determine their effect on the clonal growth of normal bone marrow (BM) cells. Twenty-one patients with AA and 11 normal controls were enrolled in this study. Medium conditioned by PBMNC of AA patients in the presence of lipopolysaccharide (LPS) was found to be suppressive to the colony growth of normal BM cells. Thus, we further determined the presence in the PBMNC-conditioned medium (CM) of both inhibitory cytokines: macrophage inflammatory protein-1 (MIP-1), tumor necrosis factor- (TNF-), transforming growth factor-2 (TGF-2), and interferon- (IFN-), and stimulatory cytokines: interleukin-3 (IL-3) and stem cell factor (SCF). Spontaneous production of MlP-1 was higher in the AA patients than the normal controls (1887±174 pg/ml vs 1643±93 pg/ml), but the difference was not significant. After LPS stimulation, the production of MIP-1 was markedly increased in the AA patients, and its level was significantly higher than that of the normal controls (2360±149 pg/ml vs 1517±92 pg/ml, p=0.0022). The level of TNF was also higher in the AA patients. However, IFN-, TGF-2, SCF, and IL-3 were not detectable in the PBMNC-CM of either AA patients or normals. The myelopoietic suppressing effect of AA-PBMNC-CM from each AA patient was significantly blocked by pretreatment with anti-TNF-, resulting in a colony-forming enhancement of 174%±12%. A similar effect was noted in six of 11 AA patients by pretreatment with anti-MIP-1. We conclude that TNF and MIP-1 can be overproduced by the PBMNC of some AA patients, which may play a role in the progression of AA.     

Influence of recombinant tumour necrosis factor a on blood flow and antibody localisation in human tumour xenografts in nude mice

Summary Mice with s.c. grafts of gastric carcinoma MKN45 or osteosarcoma 788T were injected i.v. with recombinant tumour necrosis factor (rTNF) and tumour blood flow rates were determined 4 h later as a fraction of the cardiac output g tissue^–1. With MKN45, the tumour blood flow rate was significantly reduced from a mean of 1.86% cardiac autput g^–1 to 0.84% and 0.65% with 50 and 200 ug kg^–1 rTNF respectively. With 788T, the tumour blood flow rate was reduced at 50 g kg^–1 rTNF from 1.13% cardiac output g^–1 to 0.56%. There were essentially no changes in blood flow rates in other organs. The effect of rTNF on localisation of monoclonal antibodies into these xenografts were examined. When a single dose of rTNF (50 g kg^–1) was given at the same time as labelled NCRC-2 antibody there was a significant reduction in localisation into 788T osteosarcoma xenografts. In other tests, mice were injected daily for 3 days with 50 g kg^–1 rTNF. They were injected i.v. with monoclonal antibody 4 h after the first injection and dissected on the 4th day. With 788T there was a small but not statistically significant reduction in the absolute amount of NCRC-2 antibody localising in tumour, although this reduction was greater when results were expressed as tumour-to-blood ratios. With MKN45 xenografts, treatment with rTNF had little effect on tumour localisation of an anti-(carcinoembryonic antigen) monoclonal antibody (NCRC-24). These studies show that TNF can be administered so as to reduce tumour blood flow and with little effect on tumour localisation of antibody, suggesting that combination therapy with TNF and antibodies or their immunoconjugates is feasible. Other studies have suggested that TNF can increase antibody localisation into tumours, but this was not seen here, and in some cases administration could reduce tumour localisation. It appears that this method of enhancing antibody localisation may be critically dependent on scheduling, and therefore it may not be extensively applicable.Abbreviations rTNF recombinant tumour necrosis factor - CEA carcinoembryonic antigen - BSA bovine serum albumin