The acute phase response in the rodent.

In the rodent, the general response to acute inflammation and tissue damage is characterized by a complex rearrangement in the pattern of concentrations of proteins in the plasma leading to an increase in the sedimentation rate of erythrocytes, an increase in leukocyte concentration in the bloodstream, and a decrease in the hematocrit. Body temperature changes only slightly or not at all. The reasons for the change in plasma concentrations of proteins are changes in their rates of synthesis in the liver. Degradation of plasma proteins is not affected. The details of the acute phase response evolved in the interaction of species with their environment. Therefore, it is not surprising to find differences in the details of the acute phase response among species. For example, alpha 2-macroglobulin is a strongly positive acute phase reactant in the rat, but not in the mouse; C-reactive protein is a strongly positive acute phase protein in the mouse, but is not found in the rat. An inducible acute phase cysteine proteinase inhibitor system, which has evolved from a primordial kininogen gene, has been observed so far only in the rat. The changes in the synthesis rates of acute phase proteins during inflammation are closely reflected by corresponding changes in intracellular mRNA levels. In the liver, the capacity to induce the acute phase pattern of synthesis and secretion of plasma proteins probably develops around birth. Changes in mRNA levels are brought about by changes in transcription rates or by changes in mRNA stability. Kinetics of mRNA changes during the acute phase response differ for individual proteins. The main signal compound for eliciting the acute phase response in liver seems to be interleukin-6/interferon-beta 2/hepatocyte stimulating factor, whereas interleukin-1 leads to typical acute phase changes in mRNA levels only for alpha 1-acid glycoprotein, albumin, and transthyretin. Plasma protein genes are expressed in various extrahepatic tissues, such as the choroid plexus, the yolk sac, the placenta, the seminal vesicles, and other sites. All these tissues are involved in maintaining protein homeostasis in associated extracellular compartments by synthesis and secretion of proteins. Synthesis and secretion of plasma proteins in paracompartmental organs other than the liver is not influenced by the acute phase stimuli.

mRNA for kininogens and thiostatins (major acute phase alpha 1-proteins) in the liver of kininogen-deficient rats.

mRNAs for low and high molecular weight kininogens (1.6 and 3.0 kb in size, respectively) and for two thiostatins (1.6 kb in size) were found in the liver of kininogen-deficient Brown-Norway (BN/Mai Pfd) rats. The levels of mRNAs for thiostatins, but not those for low and high molecular weight kininogens (arising from a single kininogen gene), increased strongly during acute inflammation. The pattern of DNA restriction sites for the kininogen gene and the thiostatin genes in the mutant rat strain was identical to that in at least four other rat strains.

Structure and expression of the rat transthyretin (prealbumin) gene.

The rat transthyretin gene, 7.3 kilobase pairs (kb) long, with 14.5 kb of 5' flanking and 12.2 kb of 3' flanking region was cloned and characterized. The gene contained four exons. A "TATA box" sequence (5'-TATATAA-3') and a "CAAT box" sequence (5'-GTCAAT-3') were located 23 and 95 nucleotides upstream, respectively, from the major transcription start site. Nucleotides -51 to -189 were highly conserved (93% homology between rats and humans, 97% homology between rats and mice). Tandem repeats of sequences of 5'-AC-3' and 5'-ACACATGC-3' in the 5' flanking region, of 5'-GAAA-3' in the first intron, and of 5'-GT-3' in the third intron of the gene were observed. Using specific cDNA probes, tissue specificity and regulation of transthyretin mRNA biosynthesis during embryogenesis were analyzed. Transthyretin expression occurred first in the yolk sac, then decreased when expression increased in fetal liver. Presumptive choroid plexus cells in the inner lining of the neural tube expressed transthyretin early in gestation (11 days before birth) with a maximum immediately preceding the spurt of brain growth around birth. Partial hepatectomy of adult rats induced both an acute phase response and regenerative growth in liver. The decrease in transcription of the transthyretin gene in liver, which is characteristic for the acute phase response, was overridden by stimulation of gene expression after partial hepatectomy. This stimulation also affected transthyretin expression in choroid plexus.

Structure and expression of the genes for major acute phase alpha 1-protein (thiostatin) and kininogen in the rat.

Two major acute phase alpha 1-protein (alpha 1-MAP, also called thiostatin) genes, one kininogen gene and one structurally related pseudogene, were isolated from a Buffalo rat genomic library and characterized. Comparison of the nucleotide sequence in the 5' proximal flanking region (1 kilobase) of a strongly inducible alpha 1-MAP gene with that in the non-inducible rat kininogen gene showed an overall homology of 90%, with a small number of randomly distributed base changes. In the intron downstream of the exon coding for Ile-Ser-bradykinin, the two alpha 1-MAP genes contained sequence sections similar to the section in the human kininogen gene which codes for the carboxyl-terminal chain of high molecular weight kininogen. Various features of the DNA related to gene expression such as initiation and termination sites of transcription, receptor binding sites, a Z-DNA section, and exon/intron splicing sites were identified. mRNAs expressed by alpha 1-MAP and kininogen genes in liver were studied by RNA blot hybridization using specific oligonucleotide probes. The kininogen gene expressed two mRNA species, one coding for high molecular weight kininogen, the other coding for low molecular weight kininogen. The levels of the two kininogen mRNAs in liver did not increase during acute inflammation. Only one type of mRNA, similar in size to low molecular weight kininogen RNA, was expressed by each of the two alpha 1-MAP genes. The levels of both alpha 1-MAP mRNAs increased strongly during acute inflammation.

Structure and expression of the rat apolipoprotein E gene.

The rat apolipoprotein E gene was isolated from a genomic library by screening with a cDNA probe. The nucleotide sequence of the gene plus a 5' flanking region of 623 nucleotides and a 3' flanking region of 234 nucleotides was determined. The gene contained three introns, which were located in the 5' nontranslated region and in the regions coding for the signal peptide and the mature protein. A "TATA box" sequence TATAATT was found beginning at nucleotide -32. Two copies of the sequences GGGCGG or CCGCCC, potential binding sites for the transcription protein factor Sp 1, were found adjacent to the TATA box, beginning at nucleotides -52 and -72. The 5' proximal flanking region, up to about 140 nucleotides, was found to be highly conserved (82% homology) in the gene for rat and human. This region was GC-rich (68% G + C) and contained self-complementary sequences with the potential to form hairpin loop structures, suggesting a regulatory role of this region in gene transcription. Two open reading frames with promoter sequences were found, one located in the first and the other in the second intron. Expression of rat apolipoprotein E mRNA in murine L cells transfected with the cloned gene was detected at levels comparable to that in rat liver. Rat apolipoprotein E expressed in transfected cells was secreted from the cells into the medium.

Endoscopic, histological and ultrastructural correlations in the diagnosis of colitis.

The accuracy of the endoscopic diagnosis, found at fiberoptic distal-colonoscopy, was assessed by correlation with the histological findings of multiple colo-rectal biopsies. Of 25 subjects with normal colon at endoscopy, 18 (72%) had normal histology, while 7 (28%) had histological evidence of mild non-specific colitis (6 cases) or Crohn's colitis (one case). Of 15 cases with endoscopic appearance of mild colitis, histological sections showed mild non-specific colitis in 12 (80%), idiopathic ulcerative colitis in one and Crohn's colitis in another. In 12 patients with an endoscopic diagnosis of moderate-severe colitis, histology confirmed moderate to severe idiopathic ulcerative colitis in 9 (75%) and severe Crohn's colitis in 2. Pseudomembranous colitis was confirmed by histology in 2 out of 3 cases detected by endoscopy. Electron-microscopy, although amplifying the histological findings, did not provide any specific diagnostic information. It is concluded that histological studies of multiple colo-rectal biopsies can significantly improve the diagnostic accuracy of distal-colonoscopy. Biopsies should therefore be obtained routinely in colonoscopy even if the appearances look normal.

Gastroesophageal scintigraphy and endoscopy in the diagnosis of esophageal reflux and esophagitis.

The value of gastroesophageal (G/E) scintigraphy in the diagnosis of gastroesophageal reflux was assessed in 51 subjects, who presented with heartburn and had endoscopic evidence of reflux esophagitis. G/E scintigraphy was done using 99mTc sulfur-colloid in acidified orange juice. The G/E reflux index was calculated according to previous reports. The mean (+/- SD) G/E reflux index in 18 patients with severe esophagitis and 30 patients with moderate esophagitis were 1.6% (+/- 1.5) and 3.2% (+/- 5.0), respectively. The mean G/E reflux index in 14 control subjects was 2.4% (+/- 1.1). There was no significant difference between the esophagitis and control groups. Furthermore, if 4% was taken as upper limit of normal, this will include almost all the esophagitis patients and controls. It is concluded that the G/E reflux index based on G/E scintigraphy is of little value in the diagnosis of G/E reflux.

Basal and pentagastrin stimulated 6-keto prostaglandin F1 alpha secretion in dog stomach.

The effect of pentagastrin on gastric secretion of prostacyclin (PGI2) was studied by measuring the output of 6-keto-prostaglandin F1 alpha in the gastric juice of 8 dogs before and after an intramuscular injection of pentagastrin. A similar study was done in 7 control dogs not given pentagastrin. Pentagastrin was found to significantly increase the gastric secretory output of acid and 6-keto PGF1 alpha during the first 30 minutes after the pentagastrin injection. This was highly significant when compared to the control group. It is concluded that pentagastrin stimulates gastric prostacyclin secretion.

Effective of acetylsalicylic acid, indomethacin, phenylbutazone, paracetamol, ibuprofen and naproxen on canine gastric mucosa: a histological and ultrastructural study.

The effect of various non-steroidal analgesic drugs on gastric mucosal cells was studied in 48 dogs with 2 other dogs as control. Each drug was instilled into the stomach of 8 anaesthetised dogs through a gastrostomy. Gastric biopsies, obtained at 0, 1, 2 and 3 hours after administration of the drug, were subjected to histological and electron microscopical examination. Histological analysis did not show any significant changes in gastric mucosal cells in any of the drug-related groups as compared to controls. Electron microscopy, however, showed significant changes in the gastric mucous cells and parietal cells of all test groups, as compared to controls. Ultrastructural abnormalities in mucous cells included dilation of cisternae of endoplasmic reticulum, loss of granules and microvilli, and thickening of the mucous layer and were most pronounced with acetylsalicylic acid, moderate with phenylbutazone, indomethacin and naproxen, but mild with paracetamol and ibuprofen. Parietal cell changes consisting of reduction in the concentration of tubulovesicles, which canaliculi, were found to be severe with indomethacin and phenylbutazone, moderate with paracetamol and naproxen, but only minimal with acetylsalicylic acid and ibuprofen.

P3 amylase isoenzyme in patients with cholelithiasis.

The plasma P3 amylase isoenzyme was determined in 25 patients with proven cholelithiasis. In six patients who had a single gallstone and normal pancreas at operation, the total plasma amylase was within normal limits and the P3 amylase isoenzyme was absent. In the remaining 19 patients who had multiple gallstones, pancreatitis was found at surgery in 10. In this group of 10 patients, the total plasma amylase was above the reference range for healthy individuals in only five (50%), while the P3 amylase isoenzyme band was detected in all 10 (100%). It is concluded that P3 amylase isoenzyme is superior to the total plasma amylase in the diagnosis of pancreatitis in patients with gallstone.

In vitro transformation of Syrian hamster epidermal cells by N-methyl-N'-nitro-N-nitrosoguanidine.

The selection of Syrian hamster epidermal cells which do not terminally differentiate has provided a quantitative focus assay for in vitro chemical transformation. One-day-old Syrian hamster epidermal cells plated at 5 x 10(6)/100-mm dish were treated for 5 hr with various concentrations of N-methyl-N'-nitro-N-nitrosoguanidine. After 4 weeks, the normal epidermal cells began to terminally differentiate to keratinized squamous cells and died, but transformed epidermal colonies grew to higher cells densities and appeared as darker areas against a lightly stained normal cell background. Transformed epidermal foci were isolated and subcultured for at least 15 passages, whereas normal epidermal cells could not be subcultured under the same conditions. The transformed cells assumed the typical cobblestone-like morphology of epithelial cells, retained desmosomes and tonofilaments, and were able to use citrulline in place of arginine. Argininosuccinate synthetase (EC 6.3.4.5) activity was significantly higher in the epidermal cells than in fibroblasts. The injection of 5 x 10(6) cells of two transformed epidermal cell lines into athymic nude mice resulted in the formation of tumors which were identified as keratinizing squamous carcinomas.

P3 amylase isoenzyme in the diagnosis of pancreatitis.

P3 amylase isoenzyme was found to be present in the plasma of 43 out of 140 patients with suspected pancreatitis. Of 43 patients with the P3 isoenzyme, pancreatitis was definitely confirmed in 21 by laparotomy. Of the remainder, 19 had probable pancreatitis based on a combination of clinical, biochemical, endoscopic (ERCP), ultrasonic and computerised tomographic evidence, while 3 had possible pancreatitis based on clinical evidence alone. Of 97 patients without the P3 isoenzyme 1 patient had evidence of probable pancreatitis and 1 had evidence of possible pancreatitis. Statistical analysis (excluding the 4 cases of possible pancreatitis) showed that the P3 amylase isoenzyme had a sensitivity of 98%, a specificity of 100% and an efficiency of 99%. The predictive values of a positive and negative test were 100% and 99% respectively. Hyperamylasemia was not a pre-requisite to the presence of the P3 isoenzyme, since 11 out of 13 patients, with normal total amylase levels, were found to have the P3 band and confirmation of their pancreatitis. It is concluded that the P3 amylase isoenzyme is a specific and sensitive test for pancreatitis, and appears also to be superior to the total plasma amylase.

Gluten enteropathy occurring in chronic hepatitis.

Steatorrhoea and subtotal villus atrophy due to gluten enteropathy was found to occur in a 27 year old female, who had histologically proven chronic active hepatitis six years ago. She had abnormal elevation of immunoglobulins IgG, IgM, and IgA, decreased level of complement C4 and a positive test for HLA-B8. The SGOT was persistently mildly elevated, but her latest liver biopsy (6 years after onset) showed only changes of chronic persistent hepatitis. She had good symptomatic and histological (jejunal biopsy) improvement with gluten-free diet.