Monitoring prevalence of varicella-zoster virus clades in Germany.

The global surveillance of varicella-zoster virus (VZV) clades is an important tool for investigation into viral evolution, host-virus interactions, role of immigration and travel for importation of viral strains as well as possible recombination events between wild- and vaccine-type VZV strains. In this prospective study, comprehensive data on the current distribution of VZV clades in Germany were collected. VZV strains from 213 patients with varicella and 109 with zoster were genotyped using the scattered single-nucleotide polymorphism method on the basis of sequencing open reading frames 1, 21, 22, 37, 50, 54 and 60. In varicella, clade 3 was detected in 45.5%, clade 1 in 30.0%, clade 5 in 21.1% and clade 2 in 0.9% of the cases. The analysis of zoster strains revealed clade 3 in 50.5%, clade 1 in 46.8%, clade 2 and clade 4 in 0.9% of the cases each. Five strains from varicella and one strain from zoster could not be attributed to any of the major and provisional VZV clades. Statistical analysis verified significantly lower frequency of clade 1 and significantly higher frequency of clade 5 in patients with varicella compared to zoster. In addition, varicella patients with clade 5 strains were significantly younger than the patients with clade 3. In conclusion, almost one half of VZV infections in Germany were caused currently by VZV clade 3. In primary VZV infection, nearly 20% of clade 1 has been replaced by clade 5 that might spread more effectively in the population than the European VZV clades.

Genotypes of varicella-zoster virus wild-type strains in Germany.

Surveillance of varicella-zoster virus (VZV) genotypes is indicated in Germany after implementation of universal varicella vaccination. This article reports genotyping data of 77 VZV strains obtained from 54 patients with varicella, 1 newborn with congenital varicella syndrome, 2 fetuses with intrauterine VZV infection and 20 cases with zoster. Fragments of the open reading frames (ORF) 1, 21, 22, 37, 50, 54, and 60 were analyzed by sequencing. In addition, the PstI polymorphism of the ORF 38 was characterized. Thirty strains, 22 from varicella and 8 from zoster, had the genetic markers of genotype E2, 2 of them carried new single nucleotide polymorphisms (SNP). Twenty-nine VZV isolates, 17 from varicella, and 12 from zoster, could be analyzed as E1 strains, 6 of them as E1 variants containing individual SNPs. Finally, 17 strains taken from primary VZV infection were classified as genotype M1, 13 of which belonged to the M1 subtype 1, 3 to the M1 subtype 2, and 1 to the M1 subtype 3. One strain was regarded as potential E2/J recombinant. In conclusion, VZV genotypes E2, E1, and M1 can be found in nearly equal incidence in varicella in Germany. The most frequent group is attributed to the genotype E2. Genotype M1 strains can only be detected after primary VZV infection and not in zoster cases. The possible recombinant could not be classified definitely by the scattered SNP method used and, therefore, has to be confirmed by full-genome sequencing studies.

Co-expression of GH and GHR isoforms in prostate cancer cell lines.

Prostate cancer is a significant cause of morbidity and mortality in Western males. While it is known that androgens play a central role in prostate cancer development and progression, other hormones and growth factors are also involved in prostate growth. Insulin-like growth factor-I (IGF-I) plasma levels have been associated with prostate cancer risk, and growth hormone (GH), a major factor regulating IGF levels, also appears to have a role in prostate cancer cell growth. Most significantly, GH has been shown to increase the rate of cell proliferation in prostate cancer cell lines. We have now demonstrated the co-expression of GH and GH receptor (GHR) mRNA isoforms in the ALVA41, PC3, DU145, LNCaP prostate cancer cells by reverse transcription polymerase chain reaction. Sequence analysis has confirmed that these cell lines express the pituitary form of GH mRNA and also the placental mRNA isoform. These prostate cancer cell lines also express the full-length mRNA for the GHR and the exon 3 deleted isoform. We have also demonstrated the presence of GH and GHR proteins in these cell lines by immunohistochemistry. GH expression has not been described previously in human prostate cancer cells. The co-expression of GH and its receptor would enable an autocrine-paracrine pathway to exist in the prostate that would be capable of stimulating prostate growth, either directly via the GHR or indirectly via IGF production. The GH axis in the prostate could therefore be an important additional target for the future development of prostate cancer therapies.

Transforming growth factor-alpha delays gastric emptying and small intestinal transit in suckling rats.

Transforming growth factor-alpha (TGF-alpha) is a biologically potent polypeptide detected in the gastrointestinal tract in suckling rats. The major goal of the present study was to test the hypothesis that the administration of TGF-alpha affects gastric emptying and small intestinal transit in suckling rats. Suckling (12-day-old) rats fasted 16 h received rat TGF-alpha subcutaneously (s.c.) or orogastrically in varying doses (0, 0.5, 1.0 microg/rat in 0.1% BSA). Control animals received 0.1% BSA only. Poly R-478 dye was used as a motility marker. Rats were decapitated 45 min after marker administration and the amount of dye in the stomach and the small intestine was measured by spectrophotometry. Subcutaneous administration of TGF-alpha significantly delayed stomach evacuation. In controls, the stomach contained 21.4 +/- 1.4% (mean +/- s(x)) of the Poly R-478 marker, whereas in TGF-alpha treated rats the stomach contained 37.2 +/- 2.8% of the total Poly R-478 dye given to animals. The delaying effect of TGF-alpha was time- and dose-dependent. Small intestinal transit was also significantly delayed. The proximal jejunum of TGF-alpha treated rats contained a 1.4-fold higher amount of marker in comparison with control rats. Orogastrically administered rTGF-alpha did not affect gastric emptying or intestinal transit. In conclusion, s.c. administration of rat TGF-alpha significantly delayed the gastrointestinal motility in vivo in suckling rats.

Degradation of insulin-like growth factors in small intestine of suckling rats.

Insulin-like growth factors (IFGs), IGF-I and IGF-II, present in mammalian milk, play an important role during gastrointestinal tract development. In this study we identified and localized the activities of the common intestinal proteolytic enzymes and investigated their degradation effect on IGFs. Results indicated that the enzymatic activities of chymotrypsin, trypsin, and elastase progressed from the lowest in the duodenum, to the highest in the midjejunum, and declined in the ileum. Chymotrypsin exhibited the greatest IGFs degradation activities in neonatal intestinal lumen followed by elastase. These data furnish a potential strategic design to supplement IGFs into milk formulas.

Secretion of insulin-like growth factor-II into bile of rats of different ages.

Bile from rats of different ages (suckling 10-12 days; weanling 30-33 days, and adult 60-70 days) was collected and studied for the presence of immuno- and receptor-assayable insulin-like growth factor-II (IGF-II) concentrations. Concentrations of RIA IGF-II in bile were highest in suckling rats (230 +/- 38 ng/ml) and lowest in adults (47 +/- 7 ng/ml). These concentrations were approximately twice those of the bile IGF-I concentration in sucklings, as measured in a previous study. Selected bile samples were also assayed using a competitive binding assay with a crude preparation of adult rat liver membranes bearing the IGF-II receptor. These studies confirmed the presence of receptor- (as well as immuno-) active IGF-II in bile. Since bile flow rates increased dramatically after the suckling period, bile delivery rates of IGF-II were normalized as picograms per gram body weight per hour. When such calculations were done, bile IGF-II delivery rates to the small intestine were highest in sucklings and weanlings in comparison to adult rats. Thus non-enterically derived (milk- and bile-borne) IGF-II delivery to the suckling small intestine can be approximated at roughly 1 microg/day. Unlike IGF-I, intravenously injected IGF-II could not be detected in suckling bile, suggesting a predominantly hepatic origin. From this study we conclude that there exists a significant delivery of receptor-active IGF-II to the gastrointestinal tract of rats of all ages.

Absorption of milk-borne insulin-like growth factor-I into portal blood of suckling rats.

BACKGROUND: Insulin-like growth factors (IGFs) are potent mitogens that have been implicated in control of growth and development during the perinatal period. These hormones are also present in biologically significant quantities in mammalian milks. Although one site of action of these IGFs may be at the intestinal level, current information about whether they pass intact into the circulation is conflicting. METHODS: To test the hypothesis that milk-borne IGFs are absorbed into blood in receptor-active form, suckling rats were given either recombinant human (rh)125I-IGF-I or -II (4 x 10(6) counts per minute [cpm]), and the activity present in portal and cardiac blood was examined at 5, 10, 20, and 30 minutes after ingestion for presence of appropriate molecular weight peptides in these samples. In selected samples, purified radioactive samples were tested for their ability to bind competitively to crude membranes bearing IGF receptors. RESULTS: The results of these studies indicate that rh125I-IGF-I is absorbed in receptor-active form into the portal circulation and that maximal amounts are present 20 to 30 minutes after ingestion. Estimation of the presence of intact hormone was made on the basis of the elution profile of samples when run on gel chromatography as well as reversed-phase high-performance liquid chromatography. Isolated samples from portal blood also bound competitively to placental membranes bearing IGF receptors. In contrast, rh125I-IGF-II could not be demonstrated in receptor-active form in portal blood. Chromatography showed appropriate sized peaks with greater activity in portal than cardiac samples, but competitive binding was not appreciated. CONCLUSIONS: It is likely that at least milk-borne IGF-I is absorbed intact and may exert effects on liver and other peripheral tissues. In addition, this study lends further credence to the possibility of an enterohepatic circulation for IGF-I.

Artificial formula induces precocious maturation of the small intestine of artificially reared suckling rats.

BACKGROUND: The artificially reared rat model was used successfully to study the effect of nutrition during the early postnatal period on growth and development of the neonate. Overgrowth and morphologic changes of the gastrointestinal tract are known consequences of artificial rearing. The major goal of our study was to elucidate whether artificial rearing-enhanced gut development is caused by artificial diet or by gastrostomy and the artificial rearing technique itself. METHODS: Suckling rats at day 8 of age underwent intragastric cannulation and were machine fed either a cow's milk-based artificial rat's milk substitute or pooled rat's milk for 4 days. Dam-fed littermates served as a control. RESULTS: Body growth did not differ in the three experimental groups. In rats receiving rat's milk substitute, small intestinal wet weight was approximately 60% greater than in rats fed rat's milk or control rats. Additionally, the entire small intestine was approximately 20% longer in the rat's milk substitute group. Morphologically, rat's milk substitute-fed pups demonstrated significantly greater intestinal villus length and crypt depth compared with rat's milk-fed or control rats. Jejunum and midjejunum of the rat's milk and control groups did not differ in these parameters. Intestinal sucrase activity of rat's milk substitute-fed rats was significantly elevated compared with rat's milk-fed rats or control animals. CONCLUSIONS: These results indicate that cow's milk-based formula, not gastrostomy or artificial feeding technique, is a principal cause of the small intestine overgrowth and precocious maturation of some intestinal functions observed in artificially reared sucklings.

Milk-borne epidermal growth factor modulates intestinal transforming growth factor-alpha levels in neonatal rats.

Epidermal growth factor (EGF) is present in milk from various mammalian species, but its physiologic function in neonatal development remains unclear. Transforming growth factor-alpha (TGF-alpha) is a peptide structurally related to EGF, and its presence is detected in the developing small intestine of rats. The purpose of the present study was to examine the effect of milk-borne EGF on endogenous production of EGF and TGF-alpha in the small intestine of suckling rats. Neonatal rats were fed via gastrostomy either growth factor-free rat milk substitute (RMS) or RMS supplemented with EGF (100 ng/mL of RMS) from 8 to 12 d of age. Artificially reared rats were then compared with their dam-fed littermates. Animals fed the EGF-deficient diet RMS had markedly increased EGF and TGF-alpha mRNA levels in duodenum and ileum compared with dam-fed controls and significantly elevated total intestinal content of TGF-alpha peptide. Intestinal EGF content and EGF serum levels were significantly decreased in the RMS group compared with controls. The addition of EGF to the RMS diet normalized TGF-alpha mRNA levels in the duodenum and ileum, EGF mRNA levels in the ileum, and total intestinal TGF-alpha content and EGF serum levels to the levels measured in dam-fed littermates. Motility studies showed that enteral administration of EGF did not affect stomach emptying and intestinal transit. These studies indicate that exogenous milk-borne EGF modulates endogenous production of TGF-alpha in developing small intestine. It is likely that neither TGF-alpha nor EGF are solely responsible for small intestinal overgrowth of artificially reared neonatal rats.

Luminal stability of insulin-like growth factors I and II in developing rat gastrointestinal tract.

BACKGROUND: Insulin-like growth factors (IGF)-I and -II are present in milk of a number of mammalian species. The stability of IGF-I and -II in the intestinal lumen was investigated by measuring the proteolytic degradation of 125I-labeled IGF-I and IGF-II by rat (suckling and adult) intestinal luminal flushings in vitro. METHODS: Degradation of 125I-labeled IGF-I and IGF-II was assessed by measuring the generation of acid-soluble radioactivity and the reduction of the amounts of peak activity (gel filtration). Degradation was confirmed by measuring the loss of immunoreactivity and receptor activity. RESULTS: Incubation of 125I-IGF-I with midjejunal luminal flushings from 12-day-old suckling rats generated acid-soluble radioactivity in a time- and dose-(flushing) dependent manner, whereas incubation of 125I-IGF-II produced only minor amounts of acid-soluble radioactivity. Degradation activity in luminal flushings from adult rat intestine was several times greater than that in luminal flushings from suckling rats. Degradation of 125I-IGF-II was several times lower than that of 125I-IGF-I in the intestinal luminal flushings from suckling and adult rats. The rate of decrease in immunoprecipitable 125I-IGF-I was considerably lower than the rate of decrease in receptor-active radioactivity. Except for immunoreactivity, analyses of acid-precipitable, peak-A, and receptor-active radioactivities demonstrate that IGF-II is relatively more stable than IGF-I in luminal flushings of suckling rat duodenum, jejunum, and midjejunum. CONCLUSIONS: These results show that the stability of IGF in the gastrointestinal lumen depends on the age of the animal and the segment of the gastrointestinal tract, as well as on the peptide isoform.

Human milk as a potential enteral source of erythropoietin.

In addition to its content of traditional nutrients, milk is a rich source of hormones and peptides, which survive digestion in the neonatal gastrointestinal tract secondary to lower proteolytic activity and increased protein permeability. Previous studies have shown accelerated erythropoiesis or elevated serum erythropoietin (Epo) levels in neonatal (suckling) animals after maternal phlebotomy or maternal hypoxia exposure. We sought to determine whether significant quantities of Epo are present in human milk and whether Epo remains intact under physiologic digestion conditions. Immunoreactive Epo concentrations were determined in 409 human milk samples obtained from mothers of term and premature infants. Samples collected between birth and postpartum d 134 were divided into 11 postpartum day groups. Mean milk-borne Epo concentrations were within the normal range for plasma Epo concentrations and rose with postpartum day (F10,398 = 5.82, p < 0.0001). No differences were observed between milk collected from mothers of premature versus term infants. Estimated weekly human milk-borne Epo intakes approximated the lower range of published parenteral therapeutic doses. In simulated digestion at physiologic pH levels of 3.2, 5.8, and 7.4, milk-borne Epo resisted degradation at 1 and 2 h, compared with baseline. Therefore, we conclude that human milk contains considerable amounts of Epo which resist degradation after exposure to gastric juices at physiologic pH levels. These results support continued investigation into the fate and developmental roles of Epo in human milk.

Growth of artificially fed infant rats: effect of supplementation with insulin-like growth factor I.

Insulin-like growth factor I (IGF-I), a potent mitogenic peptide, is present in considerable quantities in most mammalian milks, but its importance for the neonate is unknown. To test the hypothesis that milk-borne IGF-I is an important factor in the regulation of neonatal growth, as well as that of the gastrointestinal tract, rat pups were fed a rat milk substitute (RMS) devoid of growth factors via gastrostomy. These animals were compared with those given RMS supplemented with recombinant human IGF-I added at a concentration of 500 ng/ml. Animals given RMS + IGF-I gained mere weight than controls, although skeletal growth as represented by elongation of the tail was no different. Animals fed RMS + IGF-I had increased brain and liver wet weights as well as increased liver and small intestine protein contents. Serum IGF-I concentrations in the IGF-I-supplemented group were more than twofold above RMS controls and were similar to dam-fed rat pups. Semiquantification of serum IGF-binding proteins (IGFBP) in these animals documented that in IGF-I-supplemented pups the amount of 38- to 40-kDa molecular mass IGFBP species was also greater than in RMS controls. The rate of migration of enterocytes from crypts in duodenum and proximal jejunum was greater in IGF-I-supplemented animals than in rats fed RMS alone. These studies suggest that milk-borne IGF-I is important in modulation of somatic and gastrointestinal tract growth in the neonatal rat.

Organ distribution and biliary excretion of intravenously injected insulin-like growth factor-I in suckling rats.

The organ distribution of intravenously injected 125I-IGF-I at a dose of 2-4 x 10(6) cpm or 5-10 ng/animal was studied in 10- to 12-day-old Sprague-Dawley rats at 5 and 30 min after injection. Results of the study suggest that, although the main portion of intravenous IGF-I remains in the circulation, significant amounts are also found in the carcass, liver and kidney. Blood radioactivity fell by 50% 30 min after injection, but concentrations in the carcass, liver, kidney and skin either remained stable or increased. Gel chromatography demonstrated that significant portions of radioactivity recovered from serum, liver and kidney coeluted in a position identical to the injected IGF-I. In addition, the extracted peptide bound competitively to a membrane IGF-I receptor preparation. Studies performed on liver and kidney from these animals 5 min after injection showed that on a per gram wet weight basis, these organs contained equivalent amounts of 125I-IGF-I. However, although by 30 min, 65% of the intact labelled IGF-I has been removed from the liver, the amount remaining in kidney tissue was equal to that noted 5 min after injection. Bile was collected over a 2-hour period and contained approximately 2% of the injected radioactivity and a significant portion (30%) of this radioactivity coeluted with native IGF-I. This material also bound competitively in a radioreceptor assay, suggesting 'intactness' of this peptide. From this study, we conclude that (a) IGF-I, when administered intravenously, remains for at least 30 min in a receptor-active form in blood and several organs; (b) IGF-I derived from the circulation is cleared from the liver more quickly than from the kidney of suckling rats, and (c) that IGF-I is transferred from blood to bile.

Insulin-like growth factor-I (IGF-I) mRNA in the small intestine of suckling and adult rats.

The presence and cellular localization of insulin-like growth factor-I (IGF-I) mRNA in the small intestine of suckling and adult rats was studied. A sensitive reverse transcription (RT) competitive-polymerase chain reaction (PCR) revealed IGF-I gene expression in both age groups. Adult tissue contained 3-fold higher levels of IGF-I mRNA in comparison with sucklings. Using an in situ hybridization technique, IGF-I transcripts were localized mainly in enterocytes and goblet cells in the intestinal crypts of adult rats. By using this technique, IGF-I mRNA was not detected in jejunum of 12-day-old rats.

Insulin like growth factors and their importance in pediatrics.

Insulin-like growth factors (IGF's) are mitogenic peptides bearing homology to proinsulin. IGF's are synthesized by many cell types in mammals and have been implicated as major factors controlling growth and differentiation during fetal life, infancy and childhood. Experimental evidence for these assertions is presented, using data from animal and human studies. The effects of IGF's on normal and abnormal growth processes are discussed, and specific examples are presented of disorders related to abnormal IGF production encountered in pediatric-age patients. Discussion is also included regarding the role of milk-borne IGF for the suckling. Lastly, future uses for IGF's are mentioned, with specific examples given for treating disorders, such as short stature and diabetes due to insulin resistance.

The relation between pulmonary hypoplasia and amniotic fluid volume: lessons learned from discordant urinary tract anomalies in monoamniotic twins.

BACKGROUND: Adequate amniotic fluid (AF) volume is one of several factors felt to be essential for normal lung development. Renal agenesis and urinary tract obstruction usually result in oligohydramnios and pulmonary hypoplasia. CASE: Two sets of monoamiotic twins with discordant urinary tract anomalies were seen. One twin in each set had anomalies that in a singleton or diamiotic pregnancy would likely have resulted in fetal pulmonary hypoplasia and subsequent death. However, neither of these infants had pulmonary hypoplasia. One infant is unique in being the first case reported of normal pulmonary function and survival despite the anomaly. CONCLUSION: Adequate AF provided by a monoamniotic twin environment may prevent pulmonary hypoplasia, which usually results from oligohydramnios due to certain fetal urinary tract anomalies.

Fate of insulin-like growth factors I and II administered orogastrically to suckling rats.

Milk-borne insulin-like growth factors I and II (IGF-I and -II) may be of importance in the differentiation of the gastrointestinal tract of the suckling. To test this hypothesis, 10- to 11-d-old suckling rats were given via an orogastric tube 125I-IGF-I (n = 6) or 125I-IGF-II (n = 6) in rat milk and killed 30 min later. The results of this study demonstrated that approximately 40% of the radioactivity administered was detected in the gastrointestinal tract for both 125I-IGF-I and 125I-IGF-II experiments. Gel chromatography of acid extracts of homogenates of gastrointestinal tissues and luminal contents demonstrated that a significant fraction of recovered radioactivity eluted in a position identical to "native" IGF. These findings were confirmed by subjecting similarly treated samples to high performance liquid chromatography. In addition, radioactive material recovered from M(r) 7,500 fractions bound specifically to crude membrane IGF-I and -II receptor preparations, further suggesting the preservation of biologic activity of the recovered peptides. Although skin homogenates contained large peptide fragments of 125I-IGF-I, no "intact" IGF was found in the blood or other tissues. These findings suggest that milk-borne IGFs are stable in the neonatal gastrointestinal tract and remain biologically active for as long as 30 min postingestion.

Presence of insulin-like growth factor I but absence of the binding proteins in the bile of rats.

Whereas insulin-like growth factor I (IGF-I) has been found in various body fluids from different species, the presence or absence of IGF and associated binding proteins (IGFBPs) in bile has not been clearly defined. Bile concentration of IGF-I was measured in this study and found to be highest in the neonate and lowest in adult rats [133 +/- 15.9, 79.4 +/- 10.5, 45.3 +/- 12.7 ng/ml (mean +/- SE) in 12-day-old, 33-day-old, and adult rats, respectively]. When bile delivery rates of IGF-I (i.e., the product of IGF-I concentration in bile and the biliary flow rate) were calculated, IGF-I delivery was highest in weanling rats (469 pg.h-1.g body wt-1). When expressed as amount of IGF-I in bile delivered per day, however, delivery rates rose from 0.2 micrograms/day in the suckling and remained constant at 1.6-1.7 micrograms/day in both weanling and adult animals. Bile samples exposed to a placental membrane IGF receptor preparation showed significant dose-dependent inhibition of binding of native IGF-I. Because no IGF binding proteins were identified by Western ligand blot or by Sephadex gel chromatography, the results suggest the presence of biologically significant quantities of bioactive IGF-I in bile. We speculate that IGF-I in bile may play an important role in the growth of the gastrointestinal tract, both in the suckling as well as later in life.