Inborn errors of bile acid metabolism

Summary Cholesterol is converted to cholic acid and chenodeoxycholic acid by a series of reactions involving modifications to the steroid nucleus and oxidation of the side chain. These reactions can be affected by a number of inborn errors of metabolism. When this happens unusual bile acids or bile alcohols are synthesized; these can be identified using gas chromatography-mass spectrometry and fast atom bombardment mass spectrometry techniques. Two defects affecting the modifications to the steroid nucleus have been described; both present with cholestatic liver disease of neonatal onset. The better characterized of the two — 3-hydroxy-5-C27-steroid dehydrogenase deficiency — leads to excretion of 3-7-dihydroxy-5-cholenoic acid and 3,7,12-trihydroxy-5-cholenoic acid in the urine. The liver disease improves dramatically on treatment with chenodeoxycholic acid. Deficient activity of 3-oxo-4-steroid 5-reductase is thought to be the cause of familial liver disease in some infants who excrete 7-hydroxy-3-oxo-4-cholenoic acid and 7,12-dihydroxy-3-oxo-4-cholenoic acid in the urine. However, diagnosis of this disorder is problematical; a similar pattern of metabolite excretion can occur as a result of liver damage caused by viruses or inborn errors of pathways unrelated to bile acid synthesis. Defective side chain oxidation in patients with cerebrotendinous xanthomatosis (CTX) leads to synthesis of bile alcohols such as 5-cholestane-3,7,12,25-tetrol and 5-cholestane-3,7,12,23,25-pentol. Patients with CTX do not have cholestatic liver disease. Their major problems (neurological disease, atherosclerosis and xanthomata) are caused by accumulation of cholestanol and cholesterol in the tissues. Bile acid precursors are probably diverted into synthesis of cholestanol. Chenodeoxycholic acid suppresses the production of abnormal metabolites from cholesterol (by inhibition of cholesterol 7-hydroxylase) and leads to improvement in the neurological disease. Defective side chain oxidation also occurs in peroxisomal disorders but this time it leads to accumulation of C27 bile acids such as 3,7,12-trihydroxy-5-cholestanoic acid (trihydroxycoprostanic acid, THCA). This compound is readily detected in the bile and plasma of patients with defects of peroxisome biogenesis. In patients with defects of a single peroxisomal-oxidation enzyme (the 3-hydroxyacyl-CoA component of the bifunctional protein or the thiolase), the major C27 bile acid in bile may be 3,7,12,24-tetrahydroxy-5-cholestanoic acid (varanic acid). In addition to the above inborn errors, others which are less well characterized undoubtedly exist, as do defects of bile acid transport across membranes.     

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.     

Pharmacological properties of α1-adrenoceptor-mediated vasoconstrictions in dog and monkey lingual arteries: Evidence for subtypes of α1-adrenoceptors

Summary We examined whether ₁-adrenoceptor-mediated vasoconstrictions were due to extra- or intracellular Ca²⁺ movements, and whether they were subdivided into ₁-adrenoceptor subtypes in dog and monkey lingual arteries. The NE-induced vasoconstriction in dog lingual arteries was mostly dependent on Ca²⁺ influx from the extracellular space, since it was readily blocked by a Ca²⁺ entry blocker, diltiazem, and the NE-induced response was attenuated approximately 90% in Ca²⁺-free solution. On the other hand, in monkey lingual arteries, diltiazem failed to depress the NE-induced dose-response curve, and the response was attenuated only about 60% in Ca²⁺-free solution. The vasoconstrictor response to 10mg caffeine in normal KHS was almost the same as that to 1 µg NE in Ca²⁺-free solution in both kinds of arteries, suggesting that ₁-adrenoceptor-mediating vasoconstrictions require a different number of sources of Ca²⁺ in different blood vessels. Pretreatment of preparations with CEC (an _1B-antagonist) significantly suppressed and shifted the dose-response curve for NE to the right in monkey lingual arteries, but it had no significant effect in dog lingual arteries. However, WB4101 (an _1A-antagonist) showed almost the same potency in blocking vasoconstrictor responses as bunazosin in both kinds of arteries (the pA₂ values were not significantly different). Moreover, responses to ME (an _1A-agonist) were blocked by diltiazem as well as by bunazosin and WB4101, while CEC had no blocking effect on ME-induced responses. It is concluded that in monkey lingual arteries there are _1B-adrenoceptors with abundant _1A-subtypes linked with extracellular Ca²⁺ influx, while there are no significant _1B-subtypes in dog lingual arteries.     

Comparison of the effects of three different treatment regimens of recombinant interferons (r-IFN α, r-IFN γ, and r-IFN α +cimetidine) in disseminated malignant melanoma

Summary A total of 30 patients with progressively growing visceral and/or cutaneous malignant melanoma metastases were entered in a prospective phase II trial comparing three different therapeutic regimens of recombinant interferons (r-IFN). The first group of 12 patients received r-IFNA 9–36 IU/day i.m. on 5 consecutive days/week. A second group of 11 patients was treated with r-IFNA and oral cimetidine, 1000 mg/day. The third group of 7 patients had i.v. infusions of r-IFN 0.25–0.5 mg/m² on 3 days/week. of the 12 r-IFNA-treated patients, 1 responded (complete response, CR), 5 patients exhibited no change (NC), 3 patients had progressive disease (PD), and 3 patients could not be evaluated after therapy. In the group treated with r-IFNA plus cimetidine 3 patients responded (1 CR, 2 partial responses) and 3 exhibited NC. The remaining patients showed PD. Treatment responses were found exclusively in patients with cutaneous and/or lymph node metastases. In contrast, none of the r-IFN-treated patients responded to therapy. Known IFN side effects of varying degrees, sometimes severe, were observed in all patients. Despite the small numbers of patients treated, our preliminary data indicate that r-IFNA therapy seems (1) to be of some therapeutic value in the treatment of cutaneous melanoma metastases, (2) to be superior to r-IFN therapy, and (3) that overall response rates improve with the addition of oral cimetidine to r-IFNA treatment.     

α1-antitrypsin deficiency and liver disease

Summary ₁-Antitrypsin (₁AT) deficiency, one of the most common lethal hereditary disorders among Caucasians, is associated with emphysema in adults, while in children it is associated with liver disease. Produced in the liver and released into the plasma, ₁AT serves as the body's major inhibitor of neutrophil elastase, a powerful proteolytic enzyme capable of degrading extracellular structural proteins. The pathogenesis of the liver disease associated with ₁AT deficiency is not as well understood, but is clearly linked to specific mutations in coding exons of the ₁AT gene, and the resulting accumulation of ₁AT within hepatocytes. At present, therapy for the liver disease associated with ₁AT deficiency is symptomatic, with liver transplantation as a last resort. New strategies are being developed to suppress the accumulation of ₁AT by transferring the normal gene into the liver.     

Demonstration of a relatively hepatoselective effect of covalent insulin dimers on glucose metabolism in dogs

Summary Insulin analogues with relatively greater effect on hepatic glucose production than peripheral glucose disposal could offer a more physiological approach to the treatment of diabetes mellitus. The fact that proinsulin exhibits this property to a minor degree may suggest that analogues with increased molecular size may be less able than insulin to obtain access to peripheral receptor sites. Covalent insulin dimers have previously been shown to possess lower hypoglycaemic potencies than predicted by their in vivo receptor binding affinities. Reduced rates of diffusion to peripheral target tissues-might be an explanation for the lower in vivo potency compared to insulin. To test the relative hepatic and peripheral effects of covalent insulin dimers, glucose clamp procedures with D-[3-³H] glucose tracer infusions were used in anaesthetised greyhounds to establish dose-response curves for rates of hepatic glucose production and glucose disposal with insulin, N^B1, N^{B 1},-suberoyl-insulin dimer, and N^B29, N^{B 29},-suberoyl-insulin dimer. With N^B1, N^{B 1},-suberoyl-insulin dimer molar potencies relative to insulin were 68%, (34–133) (mean and 95% fiducial limits), for inhibition of hepatic glucose production and 14.7%, (10.3–20.9) for glucose disposal. With N^B29,N^{B 29},-suberoyl-insulin dimer potencies were 75%, (31–184) and 2.5%, (1.5–4.3), for inhibition of hepatic glucose production and for glucose disposal, respectively. The demonstration that both dimers exhibit a significantly greater effect on glucose production than on glucose disposal supports the suggestion that analogues with increased molecular size may exhibit reduced ability to gain access to peripheral target cells.Abbreviations B1-B 1D N^B1,N^{B 1},-suberoyl-insulin dimer - B29-B 29D N^B29,N^{B 29},-suberoyl-insulin dimer - Ra hepatic glucose production rate - Rd peripheral glucose disposal rate - M_r relative molecular weight - MCR metabolic clearance rate - ANOVA analysis of variance     

Effects of 5-hydroxytryptamine and adenosine on the canine isolated cerebral resistance vessels

We investigated the effects of 5-hydroxytryptamine (5-HT), adenosine, and adenine nucleotides on the canine isolated cerebral resistance arteries ranging from 300 to 500 µm in diameter. Addition of 5-HT and 5-carboxami-dotryptamine (5-CT) produced concentration-dependent contractions in the resistance arteries. Methiothepin (10^–8 M-10^–6 M) caused a parallel shift to the right of the dose-response curves for 5-HT and 5-CT. Neither ketanserin (10^–7 M, 10^–6 M) nor [(1H, 5H)-8-methyl-8 azabicyclo [3,2,1] oct-3-y1]1H-indole-3-carboxylate hydrochloride (ICS 205–930 10^–7M, 10^–6M) caused any significant shift of the dose-response curve for 5-HT. Adenosine, ATP, ADP, and AMP caused concentration-dependent relaxations in the resistance arteries following contraction with PGF2. The adenosine analogs also caused a dose-dependent relaxation of the arterial segments contracted by PGF2. The decreasing order of potency of the agonists was 5-N-ethylcarboxamido adenosine (NECA)>adenosine L-diasteroisomers of phenylisopropyl adenosine (L-PIA). The relaxant response to NECA was competitively antagonized by 8-phenyltheophylline. The pretreatment with 3×10^–5 M N^G-monomethyl-L-arginine (L-NMMA) or 5×10^–5 M aspirin caused no significant effect on the adenosine-induced relaxation in the resistance arteries. These results suggest that 5-HT produces contraction of the canine isolated cerebral resistance arteries, which is mainly mediated via 5-HT₁-like receptors. Adenosine causes an endothelium-independent relaxation of the resistance arteries through the activation of adenosine A₂-receptors.     

A deletion of 11 bp (CD 131–134) in exon 3 of the β-globin gene produces the phenotype of inclusion body β-thalassemia

Dominant inherited -thalassemias describe those -thalassemia variants that result in a thalassemia intermediate phenotype in individuals who have inherited only a single copy of the abnormal gene. This form of thalassemia is characterized by moderately severe anemia with jaundice and splenomegaly; it is also characterized by the presence of inclusion bodies in the red blood cell precursors and has, therefore, previously been referred to as inclusion body -thalassemia. We describe a case of inclusion body -thalassemia in a 51-year-old Spanish male caused by a deletion of 11 bp (CD 131–134) in exon 3 of the -globin gene. The deletion of 11 bp in exon 3 of the -globin chain is predicted to produce an anomalous chain of 134 amino acids instead of the normal 146 with an extremely altered amino acid sequence from residues 131–134. Although this shortened variant would lead to a missing H helix, which is involved in ₁₁ contact and ₁₂ subunit interactions, the variant chain can still be bound to the heme group and acquire a secondary structure that is not suitable for the formation of stable dimers or tetramers and also less susceptible to proteolytic degradation. This is the first report of such a -thalassemia mutation.     

Differential expression of cell surface integrins on human mast cells and human basophils

Summary Integrins are multifunctional recognition molecules and are expressed on various hematopoietic cells. In the present study expression of integrins on the cell surface of human mast cells and human basophils was investigated by using monoclonal antibodies (mAbs) and indirect immunofluorescence. Human mast cells were obtained from lung (n = 5), uterus (n = 5) and skin (n = 4). Human blood basophils were obtained from normal donors (n = 2). In addition, HMC-1 cells (human mast cell line) and KU-812 cells (a basophil cell line) were analyzed. Primary mast cells were found to react with mAbs directed against the common chain of 1 integrins (CD 29), the chain of VLA-4 (CD49d) and VLA-5 (CD49e), the chain of 3 integrins (CD 61), and the chain of the vitronectin receptor (VNR) (CD 51). Mast cells were not recognized by mAbs to 2 integrins (CD 18, CD 11 a, CD 11b, CD 11c), the chain of VLA-2 (CD 49 b), and VLA-6 (CD 49 f). No differences in expression of integrins on human mast cells obtained from different organs were found. HMC-1 cells and primary mast cells expressed an almost identical pattern of integrins. Human basophils and KU-812 cells were found to react with mAbs directed against 1-integrins (CD 29, CD 49 b, CD 49 d, CD 49 e) and 2-integrins (CD 18, CD 11 a, CD 11 b, CD 11 c). Together, mast cells and blood basophils express a unique pattern of integrins. These cell surface structures may be involved in the distribution of basophils and tissue mast cells and their accumulation and function in inflammed tissues.Supported by theFonds zur Förderung der Wissenschaftlichen Forschung in Österreich, grant no. P-7891     

Mutation of the 97-197-197-1subunit of the pyruvate dehydrogenase complex,in relation to heterogeneity

Summary Pyruvate dehydrogenase complex was studied using bio- and immunochemical methods in cultured cells derived from two patients with the severe type and one patient with the mild type of pyruvate dehydrogenase complex deficiency. In patients 1 and 2, enzyme activity was all but undetectable and associated with the absence of E₁ subunit of the complex. Patient 3 had a slightly reduced level of enzyme activity, and this was associated with a larger form of E₁ subunit. The amount and size of E₁ mRNA in the three patients was similar to that of control samples. Thus, the severity of E₁ deficiency in these three patients is likely to depend on the type of mutation in the pyruvate dehydrogenase E₁ subunit and the synthesis and degradation rate of the subunit.     

Mutation of E1α gene in a female patient with pyruvate dehydrogenase deficiency due to rapid degradation of E1 protein

Summary A mutation of an insertion of 4 bp in the gene for the subunit of pyruvate dehydrogenase (E1) was found in a female with pyruvate dehydrogenase deficiency due to the rapid degradation of and subunit proteins of pyruvate dehydrogenase. This mutation caused a frameshift that altered the amino acid sequence and created a premature stop codon. This 4-bp insertion has been found in an unrelated female patient with E1 deficiency. It is rare that the same mutation is found in unrelated patients with this rare inborn error of metabolism. Furthermore, short deletions or duplications in the E1 gene of patients with E1 deficiency have been found only in exons 10 and 11. These exons may be hot spots for the mutations by the recombinational processes. This patient was heterozygous for the normal and a mutant allele. However, in most of the cultured skin fibroblasts from this patient, the mutant allele was expressed. These observations suggest that the X chromosome containing the normal allele was predominantly inactivated so that she developed lactic acidaemia and neurological abnormalities despite being heterozygous. The mutant subunit protein failed to form a stable structure of pyruvate dehydrogenase, so that both and subunit proteins were degraded rapidly.     

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      

AST/ALT Ratio ≥1 Is Not Diagnostic of Cirrhosis in Patients with Chronic Hepatitis C

Medical guidelines forinterferon-_2a or-_2b(IFN-) treatment of chronic hepatitis C virus(HCV) infection depend upon baseline liver histology. Abetter long-term response to IFN- therapy correlates with less inflammation and absenceof cirrhosis. It has been suggested that the presence ofcirrhosis in patients with chronic hepatitis C virusinfection may be predicted based on an AST/ALT ratio 1. This study was designed todetermine if the presence of cirrhosis can be predictedin patients with chronic HCV infection by such a ratio.Seventyseven patients, including 23 cirrhotics, withchronic HCV infection were studied. Serum ALT, AST, andHCV-RNA levels and hepatic activity index (HAI),reflecting histologic inflammation in all liverbiopsies, were assessed. AST/ALT ratios and mean ALT,AST, and HCV-RNA were determined for both cirrhoticand noncirrhotic patients. HAI was correlated with ALT,AST, and HCV-RNA levels, the latter determined byquantitative RT-PCR. The likelihood ratio (LR) and positive predictive value of an AST/ALT ratio1 for cirrhosis was 7.3 and only 77% , respectively.In cirrhotics vs noncirrhotics, there were nosignificant differences between mean serum ALT (149± 28 vs 176 ± 17 units/liter), AST (139± 28 vs 102 ± 8 units/liter), or HCV-RNAlevels (589,160 ± 147,053 vs 543,915 ±75,497 copies/ml), respectively. There was asignificant, but clinically weak, correlation between serum ALT and HAI (r =0.234), and none between HAI and either serum AST orHCV-RNA levels. Our results support the need for a liverbiopsy prior to treatment of chronic HCV infection, since the AST/ALT ratio fails to predictaccurately the presence of cirrhosis.     

Prevention of post-menopausal bone loss with 1α-hydroxy vitamin D3 a three-year prospective study

Summary An open and controlled prospective study was used to assess the preventive efficiency of 1-hydroxy vitamin D₃ (1 (OH) Vit. D₃) on post-menopausal vertebral bone loss. Of the 36 patients included in the study, 25 completed two years of treatment with 1 g/day of 1 (OH) Vit. D₃ and 500 mg of calcium. The vertebral bone mineral density measured by dual photon absorptiometry did not vary in the treated group, whereas it decreased significantly in the control group at the end of the 2 years. At two years, withdrawal of treatment led to a signficant bone loss, whereas bone mass remained stable in a subgroup of patients who underwent a third year of treatment with 1 (OH) Vit. D₃. Overall, tolerance was satisfactory. However, urinary calcium increased significantly during treatment and one third of the patients developed hypercalciuria 7.5 mmoles/24 h. No variation in either serum calcium or creatinine levels was noted. These results indicate that 1 (OH) Vit. D₃ could be useful in preventing post-menopausal bone loss provided it was complemented by regular monitoring of urinary calcium excretion.     

Heterophilic interactions between cell adhesion molecule L1 and α<Subscript>v</Subscript> β<Subscript>3</Subscript>-integrin induce HUVEC process extension <Emphasis Type="Italic">in vitro</Emphasis> and angiogenesis <Emphasis Type="Italic">in vivo</Emphasis>

Cell adhesion molecule L1 was implicated in angiogenic processes, tumor formation and metastasis. Here, we provide evidence that the sixth Ig-like domain of L1 (L1Ig6) interacts with _{v 3} to induce process extension of human umbilical vein endothelial cells (HUVECs) in vitro and angiogenesis in vivo. HUVECs formed network-like structures on full-length L1 or L1Ig6 substrates comparable to structures found on matrigel. In the presence of mab _{v 3} or cyclic RGD, apoptosis was induced. In fibrin matrices where L1Ig6 was covalently incorporated, HUVECs formed multicellular and hollow processes through interactions between cell-surface _{v 3} and RGD-sites of matrix-immobilized L1Ig6. No such processes were induced by L1Ig6 having non-functional RDG-sites, or in the presence of mab _{v 3} or cyclic RGD. In those matrices, increased apoptosis was found. Co-immunoprecipitation of L1 or L1Ig6 with _{v 3} suggests close interactions. Furthermore, L1Ig6 stimulated HUVECs showed increased tyrosine phosphorylation of _{v 3} and phosphorylation of MAP kinases (ERK1 and ERK2) but not AKT indicating specific activation of _v and _{v 3} followed by activation of downstream kinases. Application of L1Ig6-modified fibrin matrices on CAMs induced 50–60% increased _v and _v3 protein expression and in vivo angiogenesis indicated by ~50% increased mean vascular length density. The results demonstrate angiogenic potential of L1Ig6 involving ligation and activation of _v3     

Identification ofN-acetyl-α-aminoadipic acid in the urine of a patient with α-aminoadipic and α-ketoadipic aciduria

Summary A 3.5-year-old Japanese boy with a mild speech disturbance excreted a large amount of -aminoadipic acid into the urine. The amino acid analysis using an amino acid analyser confirmed the presence of -aminoadipic acid in both urine and plasma.We detected -aminoadipic acid in the hydrolysate of the effluent and washwater fraction through cation exchange resin. This suggested the presence of acetylated derivatives and we identifiedN-acetyl--aminoadipic acid using liquid chromatography-atmospheric pressure ionization mass spectrometry (LC-API-MS). The concentrations of -aminoadipic acid andN-acetyl--aminoadipic acid in the urine of a patient with -aminoadipic aciduria were 376.9 nmol/mg creatinine and 18.1 nmol/mg creatinine, respectively. We also detected -ketoadipic acid in the urine of this patient using LC-API-MS.     

Invasive potentials of gastric carcinoma cell lines: Role of α2 and α6 integrins in invasion

The potentials of the two major histological types of gastric carcinoma to invade through extracellular matrices were studied with cell lines. We found that the invasive potential of intestinal-type carcinoma cells (MKN-28 and MKN-74) were higher than those of diffuse-type carcinoma cells (MKN-45 and KATO-III). To investigate whether the ₂ and ₆ integrin adhesion molecules are responsible for, or involved in carcinoma invasion, we further studied ₂ and ₆ expression patterns in these two types of cell line. Although fluorescence-activated cell sorting analysis revealed that all cells examined invariably expressed these integrin molecules, their expressional patterns were different among different cell lines. The intestinal-type carcinoma cells expressed integrins mainly along the cell-cell contact region, whereas the diffuse-type carcinoma cells showed a diffuse cytoplasmic pattern of integrin expression. Invasion by MKN-28, MKN-74 and MKN-45 cells through reconstituted basement membrane or type I collagen gel was significantly inhibited (P<0.05) by 50 g/ml anti-(₂ integrin) or anti-(₆ integrin) monoclonal antibodies. Our results suggest that active invasiveness is stronger in the intestinal-type than in the diffuse-type carcinoma cells and that ₂ and ₆ integrins play important roles in invasion of both types of gastric carcinoma cell lines.     

Biochemical and clinical studies of a new case of α-aminoadipic aciduria

A mentally retarded, 10-year-old female with obesity, hypotonia, clumsiness and mild ocular abnormalities excreted in her urine large amounts of -aminoadipic acid. Amino acid analyser studies and gas-liquid chromatography-mass spectrometry (GC-MS) confirmed the presence of -aminoadipic acid in both urine and plasma but, in contrast to most other patients with this disorder, failed to demonstrate significant levels of -ketoadipic acid in urine. Other known causes of -aminoadipic aciduria were eliminated by showing that levels of lysine, saccharopine and pipecolic acid in plasma and urine were normal and that the activity of glutaryl-CoA dehydrogenase was also normal.Loading with L-lysine and L-tryptophan both increased the concentration of -aminoadipic acid in blood and urine compatible with a primary deficiency of -ketoadipate dehydrogenase, in spite of the absence of -ketoadipic aciduria. Dietary restriction of lysine and administration of vitamins B₁ and B₆ were unsuccessful in correcting the biochemical abnormality.     

Cooperative effects of gamma-interferon and 1α, 25-dihydroxyvitamin D3 on in vitro differentiation of the blast cells of RAEB and RAEB-T

Summary We added recombinant human gammainterferon (-IFN) and 1 , 25-dihydroxyvitamin D₃ (1 , 25 (OH)₂D₃) to the bone marrow cells from six patients with RAEB or RAEB-T in liquid suspension cultures. After cultivation for 7 to 9 days, numerical, morphological and functional changes of the cells were assessed. -IFN and 1 , 25 (OH)₂D₃ additively suppressed cell growth, especially the number of blast cells decreased. The expression of -naphthylbutyrate esterase (NBE) activity appeared to be promoted but that of naphthol AS-D chloroacetate esterase (NAE) activity was apparently suppressed by the addition of -IFN and/or 1 , 25 (OH)₂D₃. The percentage of NBT reduction-positive cells and latex-phagocytizing cells was only slightly increased by both agents. These results indicate that -IFN and 1 , 25 (OH)₂D₃ cooperate to induce monocytoid differentiation of the patients' blast cells. Combination therapy with both agents merits further study.     

The importance of granulocyte elastase in haematological diagnosis

Summary PMN elastase is a useful additional parameter in the differential diagnosis of the leukaemias. In all patients with myelocytic leukaemias there were elevated levels of elastase-₁-proteinase inhibitor (E-₁PI), while in the lymphatic leukaemias complexed elastase levels were decreased. The highest values were found in the peripheral blood plasma and bone marrow plasma of patients with CML. Despite high E-₁PI concentrations there were no signs of bleeding or consumption of plasmatic coagulation factors. In AML a wide range of E-₁PI levels was observed, extending from slightly elevated to four hundred-fold increased. In myeloblastic leukaemias without maturation (FAB M1) the concentrations of complexed elastase remained below 150 ng/ml. In myeloblastic leukaemias with maturation (FAB M2) the E-₁PI values ranged between 214 ng/ml and 850 ng/ml (x= 402 ± 69), and in myelo-monoblastic leukaemias (FAB M4) between 450 ng/ml and 720 ng/ml (x = 663 ± 72). The only case of promyelocytic leukaemia (FAB M3) exhibited an extremely high value of 4,550 ng/ml, while a monocytic leukaemia (FAB M5) showed an extremely low value of 5 ng/ml. During cytostatic therapy there was a rapid decrease in levels of complexed elastase, with E-₁PI values returning to normal in remission. In recidivating cases there was an increase of E-₁PI levels in AML and a decrease in ALL. There was a correlation between the E-₁PI concentrations in peripheral plasma and leukaemic bone marrow infiltration, so providing a good basis for monitoring remission from leukaemia and indicating relapse. It was also interesting to observe an extremely low E-₁PI level (5 ng/ml) in patients with myelodysplasia. Under Decortin/Plenastril therapy the concentration rose to 50 ng/ml. An E-₁PI level of 10 ng per ml was observed in one case of Ranitidine agranulocytosis. Under corticoid therapy the value returned to normal within eight days.