Gastrointestinal inflammation and the circulating IGF system in humans.

Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) have important anabolic functions in normal tissue growth, which in excess may lead to tumorigenesis. In the present study, circulating IGF-I, IGF-II and their binding proteins (IGFBP-3, IGFBP-2 and IGFBP-1) were determined in 92 adult patients with gastrointestinal inflammation (Crohn's disease, colitis ulcerosa, gastritis, duodenitis errosiva, gastrointestinal candidiasis, and rotaviral and adenoviral enteritis). Serum IGF concentrations were measured by radioimmunoassay, while IGFBP profiles and IGFBP proteolytic patterns were characterized by immunoblotting. Concentrations of both IGF-I and IGF-II were significantly (p < 0.001) lower in patients than in healthy subjects. Immunoblotting demonstrated a decreased amount of intact IGFBP-3 (by approximately 60%), whereas IGFBP-2 and IGFBP-1 were increased (approximately 1.7 and 3.5-fold, respectively). No alteration in either fragmentation pattern or relative degree of proteolysis was detected in patients compared to the control group. It may be concluded that the IGF system is seriously imbalanced in patients with gastrointestinal inflammation, regardless of primary cause. These findings may help towards a better understanding of the metabolic outcome of the inflammatory process, and possibly in predicting the efficiency of patient recovery.

Periparturient endocrine and metabolic changes in healthy cows and in cows affected by mastitis.

Transition from pregnancy to lactation in dairy cows involves considerable metabolic adaptation. Additional stress is incurred during infections such as periparturient mastitis. Multiparous Holstein-Friesian cows kept under normal production conditions (n = 15) were used to evaluate changes in circulating metabolite and hormone concentrations from 5 days before to 5 days after calving. Insulin-like growth factor binding protein (IGFBP) profiles were also monitored. Marked time-related changes were observed for plasma thyroid hormone, IGF, cortisol, insulin, beta-hydroxybutyrate (BHB) and non-esterified fatty acid (NEFA) concentrations but not for plasma leptin. A decrease in IGF-II concentration and maximal intensity of the putative IGFBP-1 band occurred at parturition. When compared with the five healthy cows,low IGF-II levels were prolonged to day 2 post-partum in five cows with Escherichia coli-associated mastitis. However, marked decreases in IGFBP-2 band intensity were evident only in two of the four cases examined. Individual total ligand (IGF-I + IGF-II) concentration and IGFBP pattern prepartum were largely regained 5 days post-partum in all cows. Hormone and metabolite concentrations in the two cows with Staphylococcus aureus-associated mastitis were very similar to those in the five healthy cows. Plasma thyroxine (T4) was lower 2 days prepartum in the cows, which later developed Gram-negative mastitis. Multiregression analysis showed that variance in T4 concentration was significantly and independently associated with triiodothyronine (T3) and IGF-I positively and with cortisol negatively (R2 = 0.648). This study confirms the close inter-relationship between the thyroid hormone and IGF axes in cattle and indicates possible effects of Gram-negative mastitis infection on IGF-II metabolism.

Peripheral circulating insulin-like growth factor-I and -II in cattle.

Interrelationships between circulating concentrations of the insulin-like growth factors (IGF-I and IGF-II) were investigated in 235 blood samples taken from 145 healthy beef or dairy calves, bulls and cows of different breeds and ages. Autoradiography of Western ligand blots indicated different IGF binding protein (IGFBP) profiles between sera from different categories of cattle. Each IGF radioimmunoassay was validated by determining the effects of IGFBPs, ligand and contraligand, as well as serial dilution and comparison with results obtained after molecular sieve chromatography in acid. In female cattle mean values for IGF-I varied from 5.1 nmol/l in postparturient Holstein cows to 18.5-20.5 nmol/l in growing beef heifers, while mean IGF-II concentrations ranged from 30.0 nmol/l in the cows to 14.7-15.7 nmol/l in the beef heifer calves. In male cattle mean serum IGF-I ranged widely from 8.2 nmol/l in 1-day-old Holstein calves to 67.4 nmol/l in 16-month-old Simmental-type bulls. Mean IGF-II concentrations decreased from 22.9 nmol/l in 1-day-old Holstein bull calves to 11.9 nmol/l in 12-month-old beef bulls. Thus, total molar IGF concentrations were fairly stable in female cattle (24.7-35.1 nmol/l) but extended from 27.3 nmol/l to 81.8 nmol/l in the male cattle. The tendency for a reciprocal relationship between serum concentrations of these growth factors was most obvious in the periparturient cows.

Alterations of IGF-binding proteins in patients with alcoholic liver cirrhosis.

The protein synthetic activity of the liver is diminished in cirrhosis. The aim of this study was to investigate possible changes in the serum IGF-IGFBP system among patients with alcoholic liver cirrhosis (ALC). The results obtained demonstrated that serum IGF-I and IGF-II concentrations were significantly lower in patients with ALC than in healthy persons (P=0.0008 for IGF-I and 0.0002 for IGF-II). The IGFBP profile was markedly altered and the 34 kDa IGFBP from patients had higher affinity towards 125I-IGF-II compared to the 34 kDa IGFBP of control individuals. Moreover, the 40-45 kDa IGFBP (in isolated complex with 125I-IGF-II) exhibited diminished interaction with concanavalin A, wheat germ, and breadfruit lectins. Modification of the glyco-component of the 40-45 kDa IGFBP seems to be an early event in ALC since change in reactivity towards lectins was noticed in patients with ALC classified as Child score A, whose serum IGF-I and IGF-II levels were within reference limits (the existence of carbohydrate microheterogeneity of this IGFBP was also assessed by lectin-affinity electrophoresis). It is possible that these biochemical alterations may affect the functional activity of the IGFs by changing the dynamics and distribution of these growth factors in the organism.

Serum insulin-like growth factor (IGF)-II is more closely associated with liver dysfunction than is IGF-I in patients with cirrhosis.

The aim of this investigation was to determine the total concentrations of the insulin-like growth factors (IGF-I and IGF-II) in the blood serum of patients with liver cirrhosis and to evaluate their association with the condition. Cirrhosis was alcohol induced (n=27), of viral origin (n=17) or due to combined or other causes (n=21) and was moderate or severe in similar numbers of cases (Child A: n=21; Child B: n=21; Child C: n=23). While serum levels of both peptides were lower in patients than in age-matched healthy subjects (n=81), there was considerable overlap into the lower normal range for IGF-I. Moreover, no correlation between disease severity (Child score) and serum IGF-I was observed. Since a total of 78% of the results for IGF-II were outside the normal range (95% confidence interval) and serum concentrations were correlated with Child score (P=0.007), it is suggested that serum IGF-II concentrations may reflect compromised hepatic function more closely than IGF-I.

Colonic vasoactive intestinal polypeptide (VIP) in ulcerative colitis--a radioimmunoassay and immunohistochemical study.

BACKGROUND/AIMS: In this study, we present radioimmunoassay data describing the concentration of Vasoactive Intestinal Polypeptide (VIP) in both plasma and colonic biopsies, as well as immunostaining of VIPergic innervation in mucosal biopsies of normal subjects and patients with ulcerative colitis (UC). PATIENTS AND METHODS: Thirty three patients with UC and 17 healthy subjects were investigated. All UC patients suffered from active disease. Fasting circulating levels of VIP in plasma as well as tissue concentrations were measured by radioimmunoassay. For the immunohistochemistry, polyclonal antibody against VIP and the streptavidin-biotin peroxidase complex technique were carried out. RESULTS: Overall plasma VIP concentrations in the UC patients were similar to those in the controls. Significantly decreased concentrations of VIP were found in UC of rectum compared to the normal tissue. However, both plasma VIP values and tissue concentrations were found to be significantly lower in patients expressing minimal or mild active disease according to clinical activity index (AI) and histological activity index (HAI), but marked increase of plasma VIP was clear in UC patients with moderate or severe AI and HAI. There was a trend towards increased tissue concentrations of VIP in the group of patients with moderate or severe AI and HAI. Our immunohistochemical analysis of VIP fibers and nerve cell bodies revealed consistently weaker VIP-immunoreactivity in the rectum in UC patients with minimal or mild HAI. Simultaneously, in the rectal biopsies from UC patients with moderate and severe disease, the fibers in the lamina propria and ganglion cells in the submucous plexus were markedly increased in density and in degree of immunostaining. Very strong immunoreactivity was also found in inflammatory cells of the lamina propria as well as in the epithelial layer of the biopsies from UC patients with obvious disease. CONCLUSIONS: Our study shows clearly the heterogeneity in the response of VIP plasma level as well as rectum concentration and distribution in UC patients at different stages of the active disease. The possible role of VIP in the colon suggests that further studies of the alterations of this gut peptide may be useful in the understanding of UC pathophysiology.

Histocompatibility antigens in patients with hepatocellular carcinoma.

The distribution of class I (A, B, C) and class II (DR antigens) histocompatibility antigens (HLA) was examined in 82 patients with hepatocellular carcinoma (HCC) and in 147 patients with chronic liver disease as controls. The diagnosis of HCC was confirmed by histological examination of liver tissue. HLA-B15 antigen was found more frequently in the subgroup of HCC patients who were positive for HBsAG (13/36, 36.1%) compared to the control group (8/147; 5.4%) [p < 0.001, Pc < 0.05, RR = 9.8] and a HBsAg positive control subgroup (1/25, 4%) [p < 0.001, Pc < 0.05, RR = 13.6]. No other statistically significant difference was found for any of the HLA antigens examined either in HCC patients as a whole group or in the subgroups according to sex, course of illness, AFP status, alcohol consumption, liver cirrhosis or blood groups. These data are further evidence that there may be a link between hepatitis B viral (HBV) infection and HLA antigens. The association of HLA-B15 antigen and HbsAg supports the idea of some genetic control of HBV infection in the patients with HCC.

Rat pancreatic B-cells after chronic alcohol feeding. A morphometric and fine structural study.

Quantitative analysis of the light microscopic and fine structure of rat islet B-cells was carried out in chronic alcoholism. Absolute pancreatic weight and volume were similar in groups C (control) and E (ethanol), but relative pancreatic weight in group E rat was decreased. The results for fasting blood glucose and insulin levels were similar in the two groups of animals. There was a significantly reduced total pancreatic islet volume in E rats. The total number of endocrine cells both per islet and per microns2 of islet was similar in the two groups of animals. The volume density and number of B-cells per islet and per microns2 of islet were not changed in ethanol-treated rats as compared with the control. On the other hand, diameter, surface area and volume of the B-cells and their nuclei were found to be statistically significantly decreased. Histological examination revealed that islet blood vessels were dilated in alcoholic rats. Over the 4-month period of ethanol intake a significant decrease in cell profile area, nuclear profile area and volume density of cytoplasmic granules and an increase in the profile area and volume density of endoplasmic reticulum occurred. The gross histological alteration seen in most B-cells of the ethanol-treated rats was irregularity of the nuclear envelope with deep invagination and with margination of heterochromatin and many empty granules or granules without clear electron dense crystals of insulin. The present results indicate some optical and structural abnormalities of B-cells in chronic alcoholism that may be related to cell dysfunction and may contribute, at least in part, to the endocrine pancreas functional disturbance.

Determination of alpha-fetoprotein by radioimmunoassay. Improvement of the quality of a locally produced kit.

With the aim of improving the sensitivity and simplifying the analytical procedure, changes have been made in the test for determining alpha-fetoprotein (AFP) by radioimmunoassay, using a locally developed kit first produced about ten years ago. Close correlation between values obtained for serum AFP using the old and new procedures was obtained (r = 0.977), but the mean value for healthy nonpregnant adults was found to be 2.5 micrograms AFP/l with the new test. This is about half the level obtained earlier and confirms the recent findings of other authors. Besides reducing the time and equipment necessary for performing the test, the modified procedure enables more precise distinction between normal and elevated concentrations of AFP in patient serum.

Determination of alpha-fetoprotein by radioimmunoassay. Ten year experience in applying a locally produced kit.

Increased concentrations of serum alpha-fetoprotein (AFP) occur in several physiological (pregnancy) and pathological (liver regeneration, tumour formation etc.) conditions. Measurement of serum AFP levels may assist in the detection of fetal abnormalities during pregnancy, in the detection of renewed tumour growth and metastases in cases of AFP producing tumours and in monitoring some diseases of the liver. In this paper some results are given concerning the follow-up of some patients with tumours of the testis and the possibilities of determining the source of elevated serum AFP (regenerating hepatocytes, malignant liver cells, hepatic metastases from other tissues) in patients with liver diseases.

Immunological resistance to human biosynthetic insulin--effects of immunosuppression and plasmapheresis.

A 55-year-old gentleman, after being treated for a short time with a diet and with Chlorpropamide, was switched to purified porcine insulin due to ketonuria and ketoacidosis. After a year the patient developed immunological insulin resistance (mean daily insulin dose: 3.72 U/kg body weight; anti-insulin antibodies 78%). In order to lower anti-insulin antibodies human recombinant DNA insulin was introduced into further therapy. Contrary to expectations, the patient did not reduce whatsoever his anti-insulin antibodies and his daily insulin dose increased up to 5.63 U/kg body weight. Introduction of combined immunosuppressive therapy (prednisone plus azathioprine) together with plasmapheresis resulted in rapid lowering of daily insulin requirement and reduction in anti-insulin antibodies. Immunosuppressive therapy was continued with 10 mg of prednisone and a year later the patients insulin daily requirement was 0.66 U/kg BW while his antibodies were 18%. The possible causes of insulin resistance to human recombinant DNA insulin are discussed as well as the advantage of combined immunosuppressive therapy together with plasmapheresis that was used for rapid lowering of insulin daily requirement and anti-insulin antibodies titer.

[Synthesis, structure and function of alpha-fetoproteins and their importance in medicine]. / Sinteza, struktura i funkcija alfa-fetoproteina i njegov znacaj u medicini.

Alpha-fetoprotein (AFP) is a specific glycoprotein which is synthesised in the fetal liver and released into the blood stream together with the closely related protein, albumin. It has been proposed that AFP functions as a carrier of essential fatty acids to certain developing cells and as a possible immunosuppressor. In man its synthesis is under the strict and complicated control of transcription of a single gene on chromosome 4. The concentration of AFP in fetal serum is greatest at about 13 weeks gestation and then decreases up to birth. During pregnancy AFP passes into the amniotic fluid and also across the placenta, so that the concentration of AFP in maternal serum increases during pregnancy in a characteristic way. Greater than normal increases may indicate certain pathological states in the fetus. Serum concentrations of AFP in the newborn infant decrease rapidly to reach levels typical for adults (< 10 micrograms/L) usually by the end of the first year. Raised concentrations of serum AFP appear in a large proportion of patients with primary hepatoma and in a smaller percentage of patients with other malignant diseases (tumours of the testis, ovary, bronchi, gastrointestinal tract). In addition, increases in serum AFP are found in other illnesses accompanied by damage to hepatocytes in the liver (hepatitis, cirrhosis etc.). Certain differences in the structure of the oligosaccharide portion of the molecule have been shown between AFP synthesized by benign or by malignant cells and between AFP synthesised by hepatocytes or by cells of endodermal origin. These differences have been used as an aid in the diagnosis of liver diseases where serum AFP is elevated. Since AFP is not strictly specific for a certain type of carcinoma, its determination is primarily used in medicine for monitoring the effects of therapy and surgery on the course of malignant conditions which initially showed increased levels of serum AFP.

Serum alpha-fetoprotein levels and microheterogeneity in patients with different liver diseases.

Serum alpha-fetoprotein levels were determined in patients (268) with liver disease. Markedly elevated concentrations (greater than 100 micrograms/l) were found in twelve patients with malignant tumours and two with cirrhosis. Molecular variants of alpha-fetoprotein were distinguished by lectin affinity chromatography of these sera. Reversible binding to concanavalin A (86 +/- 5%) and to lentil agglutinin (61 +/- 19%) conformed to expected values for primary hepatocellular carcinoma except in one patient with a metastatic carcinoma whose alpha-fetoprotein binding to concanavalin A was similar to non-liver alpha-fetoprotein (44 +/- 13%), and the two patients with cirrhosis in whom binding to lentil agglutinin was typical for benign liver disorders (less than 20%). Since low levels of serum alpha-fetoprotein and non-characteristic alpha-fetoprotein binding patterns assisted in the regrouping of eleven out of 24 patients initially thought to have primary hepatocellular carcinoma, it was concluded that alpha-fetoprotein determination and lectin affinity chromatography are helpful in distinguishing primary hepatocellular carcinoma from metastatic and benign liver diseases. Slight increases in the alpha-fetoprotein level in the presence of serum hepatitis B surface antigen indicated seven patients at risk for primary hepatocellular carcinoma who should be monitored frequently.

Degradation of maize protein in rumen contents. Influence of ammonia concentration.

1. The influence of ammonia concentration on the distribution of nitrogen derived from opaque-2 maize uniformly-labelled with 15N has been investigated during short-term in vitro incubation of bovine rumen contents. 2. Less 15N derived from maize was found in the non-protein-N (NPN) fraction during incubation without added NH3 than with added NH3, due entirely to differences in the amount of N derived from maize in the NH3 fraction. 3. From calculations based on the transfer of N derived from maize to the NPN pool and to a bacterial fraction, it was concluded that degradation of maize protein was not influenced by NH3 concentration within the examined limits. 4. The decrease in relative amount of N derived from maize in the NH3 fraction at low concentrations of NH3, together with evidence for an increased fractional turnover rate of NH3-N suggests that a deficient supply of NH3 is compensated for by increased catabolism of nitrogenous compounds derived from the rumen micro-organisms.