TGF-ß signaling in liver and gastrointestinal cancers.

Transforming Growth Factor-ß (TGF-ß) plays crucial and complex roles in liver and gastrointestinal cancers. These include a multitude of distinct functions, such as maintaining stem cell homeostasis, promoting fibrosis, immune modulating, as a tumor suppressor and paradoxically, as a tumor progressor. However, key mechanisms for the switches responsible for these distinct actions are poorly understood, and remain a challenge. The Cancer Genome Atlas (TCGA) analyses and genetically engineered mouse models now provide an integrated approach to dissect these multifaceted and context-dependent driving roles of the TGF-ß pathway. In this review, we will discuss the molecular mechanisms of TGF-ß signaling, focusing on colorectal, gastric, pancreatic, and liver cancers. Novel drugs targeting the TGF-ß pathway have been developed over the last decade, and some have been proven effective in clinical trials. A better understanding of the TGF-ß pathway may improve our ability to target it, thus providing more tools to the armamentarium against these deadly cancers.

Changes of acetylcholine and choline concentrations in cerebrospinal fluids of normal subjects and patients with dementia of Alzheimer-type.

Acetylcholine (ACh) and choline (Ch) in cerebrospinal fluid from 29 normal volunteers and 7 patients with Alzheimer-type dementia (DAT) were examined using high-performance liquid chromatography with electrochemical detector coupled with liquid cation-exchange method. In normal volunteers, ACh concentration was decreased significantly from 40-50 years and Ch concentration was increased significantly from 50-60 years. CSF from patients with DAT revealed high Ch concentration and the increase was statistically significant while ACh concentration in CSF of DAT did not show a significant difference with that of normal volunteers. This Ch augmentation may suggest a disturbance in utilization of Ch for ACh synthesis and may become an useful indicator for organic changes in central cholinergic system.

Hirschsprung's disease: a search for etiology.

In 1967, Okamoto et al suggested that the absence of ganglion cells in Hirschsprung's disease (HD) was attributable to failure of migration of neural crest cells. The earlier the arrest of migration, the longer the aganglionic segment. Since then, this hypothesis generally has been accepted. However, subsequent experiments using mouse models of intestinal aganglionosis indicate that nerve cells may reach the correct position but then fail to develop or survive. An alternative hypothesis has been proposed that the aganglionosis may be caused by failure of differentiation as a result of microenvironmental changes after the migration has occurred. Extracellular matrix proteins are recognized as important microenvironmental factors. It has been shown that enteric neurogenesis is dependent on extracellular matrices, which provide a migration pathway for neural crest-derived cells and promote the maturation of settled neural crest-derived cells. Altered distributions of extracellular matrices have been shown in human HD cases and murine HD models, suggesting the role of extracellular matrices in the pathogenesis of HD. Recent studies suggest that intestinal smooth muscle cells, target cells of enteric neurons, play an important role in guiding and influencing its own innervation. Normal maturation was inhibited in neurons cultured with smooth muscle cells of aganglionic colon in comparison to normal colon. Furthermore, it was demonstrated that levels of neurotrophic factors, crucial in the development and survival of enteric neurons, are decreased in circular muscle layers of aganglionic colon in comparison to normoganglionic colon. The smooth muscle cells of the aganglionic colon may represent an unfavorable microenvironment for neuronal development compared with the normally innervated region. Recently, markedly increased immunoreactivity of major histocompatibility complex (MHC) class II antigens and ICAM-1 was demonstrated in aganglionic bowel, suggesting the immunological mechanisms may be involved in the etiology of HD. Genetic factors have been implicated in the etiology of this condition because HD is known to occur in families and in association with some chromosomal abnormalities. Recent expansion of molecular genetics identified multiple susceptibility genes of HD, including the RET gene, the glial cell line-derived neurotrophic factor gene, the endothelin-B receptor gene, and endothelin-3 gene. Of these, inactivating mutations of the RET gene are the most frequent, occurring in 50% of familial and 15% to 20% of sporadic cases of HD. To date, despite extensive research, the exact etiology of this condition remains poorly understood. The present report describes the authors' current understanding of and recent progress in the etiology of HD.

Molecular cloning and nucleotide sequencing of organophosphorus insecticide hydrolase gene from Arthrobacter sp. strain B-5.

The organophosphorus insecticide hydrolase (OPH) gene of Arthrobacter sp. strain B-5, isolated from turf green soil was cloned into Escherichia coli JM109. Three clones, termed EpB511, EpB521 and EpB531, exhibiting OPH activity were obtained. However, these three clones showed lower OP-degrading ability than strain B-5. A 7.7-kb inserted fragment of the plasmid pB521 harbored by EpB521 was subcloned, resulting in construction of a plasmid, pB526, carrying the 2.6-kb inserted fragment with OP-degrading ability. In this sequence, an open reading frame (ORF) that encodes a 43,607 Da polypeptide composed of 415 amino acids was identified. The N-terminal amino acid sequence deduced from the nucleotide sequence was identical to that of purified OPHs. The deduced amino acid sequence was compared with the sequences in the data bank and a 58.1% amino acid identity was found with the aryldialkylphosphatase from Nocardia sp. strain B-1, an enzyme that possesses catalytic functions similar to OPH.

Reduced glial cell line-derived neurotrophic factor level in aganglionic bowel in Hirschsprung's disease.

BACKGROUND/PURPOSE: Glial cell line-derived neurotrophic factor (GDNF) is suggested to be essential for the development of the enteric nervous system. The aim of this study was to investigate GDNF protein expression in human aganglionic (AG) bowel in Hirschsprung's disease (HD) using immunofluorescence and enzyme-linked immunosorbent assay (ELISA) analysis, to further understand the pathophysiology of HD. METHODS: Colonic specimens were obtained from eight patients with HD (aged 7 days to 14 months) at the time of definite pull-through surgery, including both normoganglionic (NG) and aganglionic (AG) segments. Immunofluorescence was performed using anti-GDNF polyclonal antibody and FITC-conjugated secondary antibody on the formalin-fixed and paraffin-embedded specimens. For ELISA analysis, specimens were homogenized by adding 10 volumes of 10-mmol/L phosphate-buffered saline containing proteinase inhibitors. Centrifuged supernatant was used for the quantitative analysis using sandwich-type ELISA for human GDNF. RESULTS: There was strong GDNF immunoreactivity in the mucosal epithelium, submucosal and myenteric plexuses, and hypertrophic nerve trunks. In the mucosa in AG bowel, the number of GDNF immunoreactive epithelial cells was significantly reduced compared with NG bowel (mean +/- SD, AG/NG = 227.2 +/- 73.1/310.8 +/- 80.5 GDNF immunoreactive cells per 100 crypts, P < .05). Using ELISA, the level of GDNF in full-thickness bowel was reduced significantly in AG bowel compared with NG bowel (mean +/- SD, AG/NG = 860.2 +/- 309.8/1777.5 +/- 271.4 pg/g wet tissue, P < .001). CONCLUSION: Because GDNF is essential for the development of the enteric nervous system, our findings of reduced level of GDNF in AG bowel as well as reduced number of GDNF immunoreactive cells in the mucosa of AG bowel suggest that GDNF may play an important role in the pathogenesis of HD.

Increased insulin-like growth factor and platelet-derived growth factor system in the pyloric muscle in infantile hypertrophic pyloric stenosis.

BACKGROUND: Infantile hypertrophic pyloric stenosis (IHPS) is characterized by hypertrophy of the pyloric muscle. The etiology of IHPS is unknown. The growth of smooth muscle cells (SMC) is regulated by several growth factors. Insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF) act synergistically to stimulate SMC proliferation. The effects of IGF-I and PDGF are mediated via their receptors. METHODS: Full-thickness muscle biopsy specimens were obtained from eight IHPS patients (age range, 14 to 46 days) at pyloromyotomy and from eight age-matched controls without gastrointestinal disease at autopsy performed within 4 hours after death. Indirect three-step immunohistochemistry was performed using anti-IGF-I, IGF-I receptor alpha (IGF-IR alpha), IGF-IR beta, PDGF-BB and PDGF receptor (PDGF-R) antibodies and visualized by peroxidase staining. RESULTS: The most striking difference between tissues from IHPS patients and controls was the marked increase in IGF-I, IGF-IR alpha, IGF-IR beta and PDGF-R in the hypertrophic circular muscle layer, and, to a lesser degree, in the longitudinal muscle in pyloric stenosis. CONCLUSION: The findings suggest that the upregulated local IGF and PDGF systems may play a role in the development of pyloric muscle hypertrophy in IHPS.

Morphological changes in the enteric nervous system of the transplanted fetal rat intestine.

In this study, enteric nervous system (ENS) of the fetal intestinal grafts was examined histopathologically. Forty-four rat fetal small intestines were transplanted syngenetically into the subcutaneous region of adult rats without vascular anastomosis. Thirty-two grafts survived. They were removed 2, 4, 6, and 8 weeks after transplantation and examined using (1) H&E staining, (2) AChE and NADPH-diaphorase histochemistry, and (3) protein gene product 9.5, S-100 protein, glial fibrillary acidic protein, tyrosine hydroxylase, nerve growth factor receptor, calcitonin gene-related peptide, neuropeptide Y, vasoactive intestinal peptide, somatostatin, and substance P immunohistochemistry. The grafts were compared with the intestines of 2-, 4-, 6- and 8-week-old control rats. ENS of the grafts was different from the controls as follows: (1) tyrosine hydroxylase and neuropeptide Y were markedly reduced but present, suggesting that the extrinsic innervation was present; (2) nitric oxide-producing neurons were well preserved in grafts; (3) hyperganglionosis in the myenteric plexus was seen in 6- and 8-week grafts; (4) AChE activity was increased in the circular muscle and in the lamina propria, (5) S-100 was increased in the lamina propria in 6- and 8-week grafts, (6) calcitonin gene-related peptide was increased in 6- and 8-week grafts, (7) nerve fibers in the muscle layers ran irregularly and disorderly, and (8) hypertrophy of smooth muscle layers. Our data show that although extrinsic as well as intrinsic innervation is present in the fetal intestinal grafts, there is hyperinnervation of the intrinsic nervous system and reduced innervation of the extrinsic ENS. These morphological changes in the ENS of the fetal intestinal grafts may result in motility dysfunction.

Altered insulin-like growth factor I mRNA expression in human hypoplastic lung in congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Insulin-like growth factor I (IGF-I) is a peptide growth factor that is synthesized in many organs during human development and plays a role in the growth and differentiation of tissue. IGF-I has been shown to be produced in rat and human fetal lung and to be an important mitogen involved in lung growth and development. The cells responsible for the synthesis of IGF-I in lung in vivo have been demonstrated to be type II pneumocytes, alveolar macrophages, and mesenchymal cells. Recent studies have shown that IGF-I mRNA expression in the lung is predominant during fetal life and decreases before birth, becoming barely detectable in the neonatal lung. The aim of this study was to investigate IGF-I mRNA expression in CDH lung to understand the basis of pulmonary hypoplasia in newborns with CDH. METHODS: Lung tissue samples were obtained during autopsy from 13 patients with CDH. Nine were full-term newborns (mean age, 3.8 days), and four were stillborns. Normal lung tissue from eight sudden infant death syndrome infants (mean age, 15.3 days) acted as controls. In situ hybridization was performed on frozen sections using IGF-I-specific and digoxigenin-labeled oligonucleotide probe and visualized by nitro blue tetrazolium staining. RESULTS: In control lung, IGF-I mRNA expression was absent or weak in type II pneumocytes and alveolar macrophages. In contrast, there was strong IGF-I mRNA expression in type II pneumocytes and alveolar macrophages in hypoplastic CDH lung in newborns as well as stillborns. CONCLUSION: The findings of strong IGF-I mRNA expression in the hypoplastic lung suggest that lung hypoplasia in CDH is a persistence of fetal stage of lung development.

Heterotopic splenic autotransplantation for splenomegaly secondary to Gaucher's disease--a case of siblings.

The authors report on siblings with Gaucher's disease who underwent heterotopic splenic autotransplantation for splenomegaly. The efficacy of this treatment is discussed.

Administration of antenatal glucocorticoids prevents pulmonary artery structural changes in nitrofen-induced congenital diaphragmatic hernia in rats.

BACKGROUND/PURPOSE: The aim of this study was to investigate whether maternal administration of dexamethasone has any effect on pulmonary vasculature in nitrofen-induced experimental congenital diaphragmatic hernia (CDH) in a rat model. METHODS: A CDH model was induced in pregnant rats after administration of 100 mg nitrofen on day 9.5 of gestation. Antenatal dexamethasone, 0.25 mg/kg was given intraperitoneally on day 18.5 and 19.5 of gestation. The fetuses were divided into three groups: group I (n = 10), normal controls; group II (n = 10), nitrofen-induced CDH; group III (n = 10), nitrofen-induced CDH with maternal antenatal dexamethasone treatment. The fetuses were killed by cesarean section at term. Victorian blue van Gieson staining and immunostaining with antialpha smooth muscle actin (ASMA) were performed on lung tissue. The degree of adventitial thickness and area, and medial thickness and area were measured in pulmonary arteries by image analyzer and analyzed statistically. RESULTS: There was a significant increase in adventitial thickness and area in group II compared with group I and III (P < .01). There was also a significant increase in medial thickness in group II compared with group I and III (P < .01). The degree of adventitial thickness and area and degree of medial thickness and area were similar in controls and maternal dexamethasone-treated CDH group. CONCLUSION: This study demonstrates that antenatal maternal dexamethasone treatment prevents pulmonary artery structural changes in nitrofen-induced CDH in rats.

Intestinal pacemaker C-KIT+ cells and synapses in allied Hirschsprung's disorders.

The cause of bowel dysmotility in allied Hirschsprung's disorders (AHDs) such as hypoganglionosis (HYPG), immature ganglia (IMG) and neuronal intestinal dysplasia (NID) remains unexplained. Recent experimental studies in mice have shown that c-kit gene product positive (C-KIT+) cells are responsible for intestinal pacemaker activity and that c-kit is also closely involved in synapse formation. To further understand the pathophysiology of AHDs, the authors used immunohistochemistry to study the distribution of C-KIT+ cells and synapses in the muscle layers of normal bowel from controls (12 cases) and bowel from patients with AHDs (10 patients; mean age, 3.0 years; 5 HYPG, 3 NID, 2 IMG). Anti-human C-KIT serum and monoclonal antibody 171B5 (a novel marker of synapses) were used for visualization of C-KIT+ cells and 171B5+ synapses, respectively. In normal bowel from controls and patients with AHDs, moderate to many C-KIT immunoreactive (C-KIT-IR+) cells were observed in the muscle layers. Myenteric plexuses were clearly demarcated by C-KIT-IR+ cells. 171B5 immunoreactive (171B5-IR+) synapses were abundant in the muscle layers and within the myenteric plexuses. In contrast, the number of C-KIT-IR+ cells or 171 B5-IR+ synapses was reduced in the muscle layers of bowel affected by AHDs, except within the myenteric plexuses, where there was a moderate to large number of 171B5-IR+ synapses identified. A lack of intestinal pacemaker C-KIT+ cells may be of great significance with respect to the bowel dysmotility associated with AHDs and also to the abnormal synapse formation seen in the muscle layers of bowel affected by these disorders.

Idiopathic gastric perforation in neonates and abnormal distribution of intestinal pacemaker cells.

BACKGROUND/PURPOSE: The etiology of idiopathic gastric perforation (IGP) in neonates is unclear. Interstitial cells of Cajal (ICC) express tyrosine kinase receptor C-kit, and act as gastrointestinal pacemaker cells. Stem cell factor (SCF) is a C-kit ligand and plays an important role in immune system homeostasis in the gastrointestinal tract. The authors hypothesized that abnormal distribution of ICC or SCF in the gastric wall (ie, abnormal motility or impaired immunity) could predispose the stomach to IGP. METHODS: Stomachs obtained at postmortem from neonates who died of IGP (n = 7) and other causes (control group; n = 10) were used. Biopsy sections were taken at random from various sites in the stomach, including macroscopically intact areas, and labeled immunohistochemically using antibodies to C-kit(a marker for ICC) and SCF. RESULTS: In all control specimens, ICC were present between the muscle layers and around the myenteric plexuses of the stomach wall. In contrast, ICC were absent in all biopsy sections from 3 of the 7 IGP stomachs. In the remaining 4 IGP stomachs, there were fewer ICC in the muscle layers compared with controls, and ICC were absent around the myenteric plexuses. The distribution of SCF immunoreactivity in IGP and control specimens was similar. CONCLUSION: The findings suggest that a lack of ICC (ie, gastric hypomotility) may be implicated in the etiology of IGP in neonates.

Fetal esophageal transplantation in rats: a treatment option for long-gap esophageal atresia.

PURPOSE: The aim of this study is to determine if fetal esophageal transplantation can create viable esophageal tissue that may be used for treating long gap esophageal atresia. METHODS: Fetuses of gestational age 19 to 20 days were obtained by hysterotomy of pregnant 15-week-old Lewis rats. A 10-mm long segment of esophagus was obtained from each fetus by thoracolaparotomy and transplanted by wrapping it in a pouch created in the distal omentum of a 5-week-old Lewis rat (syngeneic transplantation: n = 15). Transplanted fetal esophageal grafts were harvested 10 days post-transplantation and fixed in 10% formalin and embedded in paraffin. H&E was used for histological examination, and PGP 9.5 (a neuronal antibody) was used for immunohistochemistry. Esophageal segments obtained from 10-day-old Lewis rats were used as controls. RESULTS: Thirteen of 15 (87%) grafts were transplanted successfully. The successfully transplanted graft could be mobilized to the thoracic cavity without tension or compromising of vascularity, because of the long omental pedicle. H&E staining and PGP 9.5 immunohistochemistry showed normal esophageal structure with intact esophageal nervous system, comparable with control specimens. CONCLUSIONS: Fetal esophageal transplantation produces viable esophageal tissue that may find application for treating long gap esophageal atresia providing rejection can be controlled adequately.

Abnormal distribution of intestinal pacemaker (C-KIT-positive) cells in an infant with chronic idiopathic intestinal pseudoobstruction.

BACKGROUND: Chronic idiopathic intestinal pseudoobstruction (CIIPO) is a rare syndrome with an obscure pathogenesis. The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor C-KIT that is critical for the development of the interstitial cells of Cajal, cells that are regarded as being the pacemaker cells of the gut. Thus, C-KIT immunopositive (C-KIT-) cells in the muscle layers of the bowel are considered to be intestinal pacemaker cells. METHODS: In this study, the distribution of intestinal pacemaker cells was examined for the first time using C-KIT immunohistochemistry in an infant with CIIPO. RESULTS: C-KIT+ cells were found lying on either side of the border between the two muscle layers (longitudinal and circular) of the bowel and dispersed unevenly throughout both muscle layers. Myenteric plexuses were not demarcated by C-KIT+ cells. In contrast, in controls, C-KIT+ cells were located distinctly between the two muscle layers of the small bowel and dispersed evenly throughout the muscle layers of the colon. Myenteric plexuses were clearly demarcated by C-KIT+ cells. CONCLUSIONS: This case demonstrates for the first time that there is abnormal distribution of intestinal pacemaker cells in CIIPO and provides new evidence that abnormal c-kit gene expression may be responsible for autonomic gut dysmotility. C-KIT immunohistochemistry may be an indispensable tool for diagnosing CIIPO.

Complications after cyst excision with hepaticoenterostomy for choledochal cysts and their surgical management in children versus adults.

The aim of this study was to review the cases 200 children and 40 adults who had cyst excision combined with hepaticoenterostomy (CEHE) for choledochal cyst, with particular emphasis on post-CEHE complications and their surgical management. Patients who had CEHE at the age of 15 years or less were defined as children, and those aged 16 years or older were defined as adults. The mean age when patients became initially symptomatic was 3 years in children and 26 years in adults. Eleven adults became symptomatic as children (< or = 15 years of age). The mean age of CEHE in children and adults was 4.2 years and 35 years, respectively. The time interval between the onset of initial symptoms and CEHE was significantly less in children than in adults (P < .0001). Of the 200 children, 176 had primary CEHE, and 24 had secondary CEHE converted from cystoenterostomy or other biliary surgery. Seventy children had intraoperative cyst endoscopy, which enabled us to examine the proximal intrahepatic bile ducts for stenosis and debris, and to wash out debris, protein plugs, and stones from the intrapancreatic ducts. Of the 40 adults, 22 had primary CEHE, 18 had secondary CEHE. The mean follow-up period was 10.9 years in children and 10.7 years in adults. The number of patients with post-CEHE complications in children and adults was 18 (9.0%) and 17 (42.5%), respectively. The post-CEHE complication rate in children was significantly lower than in adults (P < .0001). The 18 children had 25 post-CEHE complications such as cholangitis, intrahepatic bile duct stones, pancreatitis, stone formation in the intrapancreatic terminal choledochus or pancreatic duct, and bowel obstruction. Twenty-seven post-CEHE complications developed in the 17 adults including 2 cases of cholangiocarcinoma. There were no post-CEHE complications in the 70 children who had intraoperative cyst endoscopy. No stone formation was seen in the 145 children who had CEHE at the age of 5 years or less. Eight stone formations were seen in seven (12.7%) of the remaining 55 children aged over 5 years. Stones developed in seven (17.5%) adults. The incidence of post-CEHE stone formation in children aged 5 years or less was significantly lower than in other children and adults (P < .0001). Reoperation was required in 15 children: revision of hepaticoenterostomy in 4, percutaneous transhepatic cholangioscopic lithotomy (PTCSL) in 1, excision of intrapancreatic terminal choledochus in 2, endoscopic sphincterotomy of the papilla of Vater in 1, pancreaticojejunostomy in 1, and laparotomy for bowel obstruction in 6. Ten adults required reoperations: revision of hepaticoenterostomy in 2, PTCSL in 2, left hepatic lobectomy in 1, endoscopic sphincterotomy in 2, exploratory laparotomy in 2, and adhesiolysis in 1. The authors conclude that early diagnosis followed by CEHE is the treatment of choice for choledochal cyst, and intraoperative cyst endoscopy is recommended as a valuable adjunct to CEHE.

Spontaneous resolution of an acute spontaneous spinal epidural hematoma without neurological deficits.

A 46-year-old woman presented with sudden severe pain in the interscapular region. Physical examination, including detailed neurological evaluation, did not disclose any abnormalities. However, magnetic resonance imaging revealed an epidural hematoma anterior to the thoracic spinal cord and its spontaneous resolution thirty days after onset. Her hospital course was uneventful. To our knowledge, this report documents the first case of a spontaneous spinal spidural hematoma without neurological deficits. Spinal epidural hematoma may be more common than previously thought because some cases have probably been misdiagnosed as transient back pain of unknown etiology.

An autopsy case of malignant lymphoma with Lyell's syndrome.

We present a case of fatal Lyell's syndrome which developed following a CT examination using omnipaque 3000 contrast medium. A 59-year-old man was suffering from malignant lymphoma. He was readmitted to this hospital due to relapse of fever and lymph node swelling. On the day of readmission, generalized erythema, purpura, and mucosal erosions developed after a CT examination. Steroids and chemotherapy were ineffective, and he expired approximately two weeks after admission. Drug-induced dermatopathy or leukemic cell infiltration in the skin was clinically suspected. Histological findings disclosed toxic epidermal necrolysis.

A case report of dorsal pancreas agenesis diagnosed by MRI and ERCP.

We present a case of dorsal pancreas agenesis demonstrated by MR imaging. Careful observation with endoscopic retrograde cholangiopancreatography and MR imaging led to the precise diagnosis.

A septum-like structure of the gallbladder in acute viral hepatitis: CT demonstration.

We present two patients with a septum-like structure of the gallbladder on CT. The patients were hospitalized with acute viral hepatitis. CT performed within a week from the onset of symptoms demonstrated a high density linear structure in the gallbladder mimicking a septum. This structure seemed to be produced by a marked thickening of the gallbladder wall. It disappeared during the clinical recovery.

The first outbreak of fowl cholera in Muscovy ducks (Cairina moschata) in Japan.

The first outbreak of fowl cholera occurred in a flock of Muscovy ducks (Cairina moschata) in Okinawa Prefecture of Japan in November 1990. Fifty (25%) of 200 birds in a farm died of an acute disease. Remaining birds recovered after treatment with oxytetracycline. Pasteurella multocida subsp. multocida was isolated in pure culture from all tissues tested from two dead birds. Serovars of the isolates were identified as Carter's capsular type A. Heddleston's type 3.4.12, and Namioka's type 5:A which have not been demonstrated in Japan. Pathologically, multiple necrosis and bacterial aggregates were prominent in several organs, particularly in the liver. The isolate killed chickens when inoculated intravenously at a concentration of 10(8) colony forming units.