Optical Coherence Tomography Identifies Lower Labial Salivary Gland Surface Density in Cystic Fibrosis

The labial minor salivary glands (LSGs) are easily accessible mucus-secreting structures of the alimentary tract that may provide new information on the basis of gastrointestinal complications of cystic fibrosis (CF). It was shown that they are destructed in the course of cystic fibrosis. We employed wide-field, micrometer resolution in vivo optical coherence tomography to assess the surface density of LSGs in 18 patients with CF and 18 healthy subjects. The median LSGs’ surface densities in CF patients, and in the control group were 4.32 glands/cm² and 6.58 glands/cm², respectively (p = 0.006; Mann-Whitney U test). A lower LSG surface density is a previously unrecognized CF-related pathology of the alimentary tract.     

The activity of cathepsin D in saliva of cystic fibrosis patients

Cystic fibrosis (CF) is genetically determined illness, which is caused by the mutation in the CFTR gene. CFTR protein is also expressed in epithelial cells of parotid glands, therefore parotid glands are also affected in CF patients. Cathepsin D is one of the proteolitic cascade enzymes. Physiological wearing out result in occurrence of trace quantities of this enzyme in serum and body fluids, including saliva. Among different enzymes, saliva contains cathepsin D (CTSD, EC 3.4.23.5). The aim of this study was to determine cathepsin D activity in mixed saliva in cystic fibrosis patients and healthy controls. The study was performed in a group of 26 CF patients (10F, 16M). The results obtained in CF group was compared with the results of thirty healthy subjects (12F, 14M). From each subject 8 ml of mixed saliva was obtained: before and after the stimulation of saliva excretion using paraffin pledgets. Protein and glycoprotein content was assessed using Winzler's method. Protein concentration in controls and CF group before stimulation of excretion was 1.15+/-0.714 mg/mL and 1.54+/-0.925 mg/mL. After stimulation protein concentration in saliva has lowered to 0.88+/-0.77 mg/mL in CF group and 1.24+/-1.213 mg/mL in controls. Glycoprotein concentration in controls and in CF group was respectively: before stimulation 1.08+/-0.271 mg/mL and 1.05+/-0.344 mg/mL; after stimulation 0.92+/-0.292 mg/mL and 0.86+/-0.283 mg/mL. The activity of CTSD in controls was 45.9+/-24.98 Tyr nmol/mL/4h before stimulation and 109.3+/-56.94 Tyr nmol/mL/4h after stimulation of excretion. In CF group CTSD activity before stimulation was 134.5+/-81.80 Tyr nmol/mL/4h and after stimulation 134.4+/-62.18 Tyr nmol/mL/4h. Comparing the CTSD activity in both groups statistically significant difference has been revealed in samples collected before stimulation of excretion (p=0.013). The activity of cathepsin D in saliva of cystic fibrosis patient is significantly higher than in healthy controls before the stimulation of excretion with paraffin pledgets.     

Species specificity of radioreceptor assay and radioimmunoassay for rat FSH

Species specificity of the radioreceptor assay (RRA) for rat FSH, in which pregnant mare serum gonadotropin (PMSG)-treated immature rat ovary was employed as the receptor, was compared with that of NIAMDD rat FSH radioimmunoassay (RIA). In the RIA system, pituitary preparations from mammals only showed significant crossreaction. Their inhibition curves, however, were not always parallel to the standard curve. On the other hand, in the RRA system, the pituitary preparations from mammals, avians, lizard and amphibians competitively inhibited the binding of radioactive rat FSH to the ovarian receptor. Only the pituitary preparation from dog salmon failed to show any crossreaction in the RRA system. These results indicated that this RRA system would be useful for the measurement of FSH or gonadotropins of the pituitaries from mammals to amphibians.     

Altered intestinal chloride transport in cystic fibrosis

Sodium ion and chloride transport was studied in vitro in small intestinal and colonic tissue from patients with cystic fibrosis (CF) and from non-CF control subjects matched as to age and sex. Normal histological appearance and substantial response to mucosal glucose (5 mM, ileum) or mucosal amiloride (10(-5) M, colon) indicated normal tissue viability in both control and CF tissues. Electroneutral NaCl absorption was demonstrated in the small intestine of control subjects and CF patients. Small intestinal and colonic tissues of control subjects responded to four secretagogues (theophylline, 5 mM; prostaglandin E2, 10(-6) M; calcium ionophore (A23187), 10(-5) M; bethanechol, 5 x 10(-5) M), with electrogenic chloride secretion. The tissues of CF patients, however, did not respond to any of the test secretagogues. These studies demonstrate that an abnormality in chloride transport is present in the small intestinal and colonic epithelia of CF patients. Unlike airway epithelia, which secrete chloride in response to Ca ionophore, the intestinal epithelia of CF patients do not respond to either cAMP- or Ca-mediated secretagogues. This abnormality in intestinal electrolyte transport may play a role in the pathogenesis of meconium impactions in CF patients.     

Correlations of Salivary Biomarkers with Clinical Assessments in Patients with Cystic Fibrosis

Rationale

Monitoring clinical disease status in cystic fibrosis frequently requires invasive collection of clinical samples. Due to its noninvasive collection process and direct anatomic relationship with the lower airway, saliva shows great potential as a biological fluid for cystic fibrosis monitoring.

Objectives

To measure the levels of multiple protein markers in human saliva supernatants and investigate the possibility of utilizing them to provide a more quantitative measure of disease state for use in research and monitoring of patients with cystic fibrosis clinically.

Methods

Whole saliva samples were collected and processed from cystic fibrosis patients at two distinct time points (2010 and 2013) and measured by two separate platforms. In this cross sectional study, a convenience sample of 71 participants were recruited with samples measured by multiplexed fluorescence microarray (fiber microarray) and another 117 participant samples were measured by an automated, point-of-care, analyzer (SDReader) using a microsphere-based array via fluorescence sandwich immunoassay. For comparison, saliva from 56 and 50 healthy subjects were collected, respectively. The levels of six target proteins were quantified. Various demographic and clinical data, including spirometry, medical history, and clinicians’ assessments were also collected from patients with cystic fibrosis on the day of saliva collection.

Measurements and Main Results

Similar trends were observed with both platforms and compared with healthy subjects, cystic fibrosis patients had significantly elevated levels of VEGF, IP-10, IL-8, and EGF as well as lower levels of MMP-9 (P ≤ 0.005) using fiber microarray and significantly elevated levels of IP-10, IL-8 with lower levels of MMP-9 and IL-1β (P ≤ 0.02) using the SDReader. The levels of the six proteins correlated with each other significantly, and in some cases, biomarker levels could be used to differentiate between subgroups of patients with different clinical presentations. For example, IP-10 levels significantly correlated with FEV₁ and disease severity (as evaluated by clinicians) with both platforms (P < 0.05).

Conclusions

Significant variations of the levels of six proteins in saliva supernatants, and the correlations of these levels with clinical assessments, demonstrated the potential of saliva for cystic fibrosis research and monitoring.     

Exhaled carbon monoxide concentration increases after exercise in children with cystic fibrosis

Oxidative stress and hypoxia, which may occur in cystic fibrosis patients (CF) at rest and may be worsened by exercise, induce the expression of heme oxygenase (HO)-1, resulting in increased carbon monoxide (CO) formation. We tested that exhaled CO level (eCO) was higher in CF patients than in healthy subjects, and that exercise increased CO production. Exhaled CO was measured electrochemically in 15 CF patients and 15 control subjects at rest (T0), immediately (T1) and 60 minutes after a symptom-limited incremental bicycle exercise test (T60). Arterial oxygen saturation (TcO2) was monitored transcutaneously. Data are given as mean+/-SEM. Baseline eCO was 1.90+/-0.26 ppm in the control and 1.93+/-0.27 ppm in the CF group. In both groups eCO was lower at T1 than at rest. In the control group eCO was also low at T60, but in the CF group it was increased compared to baseline level at this timepoint. Exercise caused oxyhemoglobin desaturation in CF patients which was related to the increase in eCO measured at T60 (r=0.67, p<0.01). Our findings suggest that exercise modulates the level of exhaled CO partly by worsening oxygenation in CF patients.     

XV-2c/KM19 haplotypes analysis of cystic fibrosis patients from western Mexico

The analysis of polymorphic markers within or closely linked to the cystic fibrosis transmembrane regulator (CFTR) gene is useful as a molecular tool for carrier detection of known and unknown mutations. To establish the association between mutations in the CFTR gene in western Mexican cystic fibrosis (CF) patients, the distribution of XV2c/KM19 haplotypes was analyzed by PCR and restriction enzyme digestion in 384 chromosomes from 74 CF patients, their unaffected parents, and normal subjects. The haplotype analysis revealed that haplotype B was present in 71.9% of CF chromosomes compared to 0% of non-CF chromosomes. The F508del and G542X mutations were strongly associated with haplotype B (96.7% and 100% of chromosomes, respectively). The haplotype distribution of the CF chromosomes carrying other CFTR mutations had a more heterogeneous background. Our results show that haplotype B is associated with CFTR mutations. Therefore, haplotype analysis is a suitable alternate strategy for screening CF patients with a heterogeneous clinical picture from populations with a high molecular heterogeneity where carrier detection programs are not available. In addition, it may be a helpful diagnostic tool for genetic counseling and carrier detection in the relatives of CF patients and in couples who are planning to have children.     

Radioimmunoassay for human salivary amylase

A radioimmunoassay (RIA) for human salivary amylase was developed. Human salivary amylase was purified from parotid saliva by a combination of Sephadex gel filtration and cation exchange chromatography. Purified salivary amylase was used both as the standard antigen and for the generation of ¹²⁵I-labeled amylase. Antibody to salivary amylase was raised in New Zealand white rabbits and used in a nonequilibrium double-antibody procedure for the RIA. The RIA was sensitive (10 ng/ml) and specific, displaying a limited cross-reactivity for pancreatic amylase (1%, $\text{w}\text{w}$). Analysis of patient sera by RIA shows that salivary amylase constitutes approximately 60% of the total serum amylase, that the salivary amylase found in the serum of patients with Sjögren's syndrome and macroamylasemia is immunologically indistinguishable from that of normal persons, and that salivary amylase can be evaluated by RIA in the serum of patients with pancreatitis.     

Hydroxyapatite in the Pathogenesis of Cystic Fibrosis

Submandibular saliva collected from cystic fibrosis patients and control subjects was separated by centrifugation into an insoluble deposit and a clear supernatant. The resulting calcium and phosphorus analyses performed on both fractions warranted a closer investigation as a consistent Ca/P molar ratio of 1·5 was found in the deposit of the cystic fibrosis patients, while no consistent ratio >1·0 was found in the deposit of the control subjects. The expected result, that calcium and phosphorus in the deposit of cystic fibrosis patients is present as the solid phase of hydroxyapatite, was confirmed by a detailed comparison of x-ray powder diffraction patterns of an ashed sample of this deposit and a similarly treated synthetic sample of hydroxyapatite.     

Dynamic change in charge heterogeneity of pituitary FSH throughout the estrous cycle in female rats

Anterior pituitaries were removed from female rats at various stages during the estrous cycle and FSH was fractionated by isoelectric focusing (IEF). Fourteen FSH components were observed during the estrous cycle and twelve of them were distributed between pH 3.71 and 6.66. IEF profiles of FSH in the pituitaries varied with the stage in the estrous cycle. Especially at the time of serum FSH surge on the day of proestrus, most of the components decreased, while only a highly alkaline component showed an increase. When these FSH components were separated and their nature was examined by radioreceptor assay (RRA), radioimmunoassay (RIA) and gel filtration, differences were observed among these components in the RRA/RIA ratio and gel filtration profile. As a general tendency, the RRA/RIA ratio of the components became greater while the apparent molecular size became smaller, as their pI became higher. However, some highly acidic components showed a biphasic elution pattern and the most acidic one eluted the latest on gel filtration, suggesting that these components may be heterogeneous in terms of molecular size. The FSH concentration in sera collected at different stages in the estrous cycle was measured by both RRA and RIA. The RRA/RIA ratio was high when the serum immunoreactive FSH was low, and low during the FSH surge. From these findings, it is concluded that the quality of FSH molecules present in the anterior pituitary gland changes dynamically throughout the estrous cycle, especially during the period of serum FSH surge. Furthermore it is suggested that the type of FSH secreted from it also varies according to the stage in the estrous cycle.     

Detection of cystic fibrosis homozygotes and heterozygotes by serum isoelectrofocusing

Summary It is suggested that the majority of individuals with the cystic fibrosis (CF) gene have a unique serum protein (CFP) which can be demonstrated by means of isoelectrofocusing (IEF) in thin layer polyacrylamide gels. We have found the CFP in 90% of patients with cystic fibrosis, in approximately 80% of individuals heterozygous for the CF gene, and in 8% of normal control individuals. We conclude that CFP is a useful marker for the CF gene.     

An immunohistochemical study of secretagogue-induced secretion of epidermal growth factor in the submandibular salivary glands of mice

Routine histological and indirect immunofluorescence techniques were used to examine the histological details of changes in the distribution of epidermal growth factor (EGF) in the submandibular salivary glands of mice during secretion. Comparisons were made bewteen glands of normal mice and those of mice given one of a number of secretagogues at various times prior to sampling. Normal submandibular salivary glands in male mice had an extensive system of convoluted granular tubules (CGT), the cells of which contained EGF. When adrenaline of alpha-phenylephrine was administered, the CGT cells degranulated, and there was a concomitant loss of intracellular EGF-positive immunofluorescence. The excretory ducts were engorged with immunofluorescent material, indicating secretion of EGF into saliva, while the ductal cells themselves remained EGF-negative. The degranulation response could be blocked by phentolamine, but not by propranolol, and no changes in EGF distribution followed the administration of pilocarpine. It was concluded that EGF is secreted, at least partly into the saliva, following an alpha-adrenergic response, and that this occurs with degranulation of the cells of the CGT.     

Radioimmunoreactivity and receptor binding activity of specific epididymal proteins of the rat and other species

Radioimmunoassay (RIA) and radioreceptor assay (RRA) for the 32K Rat Epididymal Protein (REP) have been developed. Washed intact rat epididymal spermatozoa were used as receptors in the RRA. The effects of epididymal and seminal fluids of other species in these assays were studied. The results showed that there is species specificity in the immunoreactivity and receptor binding activity of sperm coating proteins.     

Quantitative variation in cystic fibrosis-associated proteins in cystic fibrosis patients,carriers, and controls

Summary Serum samples from patients with cystic fibrosis (CF), obligate heterozygotes, and normal controls have been examined by isoelectric focusing (IEF). Our results suggest that cystic fibrosis protein (CFP) is a normal serum protein exhibiting quantitative variation primarily dependent on possession of the CF allele. It is concluded that detection of CFP by IEF is an inappropriate screening test for the CF gene due to lack of specificity.     

Discrimination of a colony stimulating factor subclass by a specific receptor on a macrophage cell line

Utilizing the high affinity interactions between pure ¹²⁵I-L cell colony stimulating factor and its receptor(s) on the murine macrophage cell line J774, a murine radioreceptor assay (RRA) has been developed. The murine RRA selectively detects a colony stimulating factor (CSF) subclass (CSF-1) previously defined by murine radioimmunoassay (RIA) (E.R. Stanley, Proc. Nat. Acad. Sci., USA, 76:2969–2973 ('79)). CSF-1 stimulates macrophage production exclusively, and the occurrence of the CSF-1 receptor(s) appears to be restricted to cells of the mononuclear phagocytic system (L.J. Guilbert and E.R. Stanley, J. Cell Biol. 85:153–160 ('80)). The murine CSF-1 RRA failed to detect a variety of other CSF subclasses, growth factors, and hormones. In contrast to data obtained with the murine CSF-1 RIA, human CSF-1 (e.g., human urinary CSF) is detected by the mouse CSF-1 RRA almost as sensitively as murine CSF-1. In addition, there was an absolute correlation between CSF-1 levels determined by murine CSF-1 RRA and those determined by a human CSF-1 RIA for a variety of human CSF-1 sources. The murine CSF-1 RRA is a sensitive (sensitivity 5 units or 1.0 femtomole of CSF-1 protein), rapid, and highly specific assay for CSF-1 in both murine and human sources.     

大鳞大马哈鱼垂体生长激素的分离、纯化与鉴定

应用ConA-Sepharose 4B亲和层析、凝胶过滤及离子交换层析等技术从大鳞大马哈鱼(Oncorhynchus tshawytscha)垂体中分离纯化了具有生物活性的生长激素(sGH)。用放射受体测定法(RRA)检测sGH组分的生物活性,结果表明纯化的8GH制品具有与兔肝细胞GH受体结合的生物活性。用放射免疫测定法(RIA)和酶联免疫吸附测定法(ELISA)分别检测了另两种垂体激素-催乳激素(PRL)和促性腺激素(GTH)在纯化的sGH制品中的残留量均在0.5％以下。用SDS-聚丙烯酰胺凝胶电泳SDS-PAGE评价sGH制品的电泳纯度并测定了其分子量为22000左右。等电聚焦电泳表明该种鱼GH由等电点分别为6.3和6.6的两种形式的分子组成。     

Regulation of normal and cystic fibrosis airway epithelial repair processes by TNF-α after injury

Chronic infection and inflammation have been associated with progressive airway epithelial damage in patients with cystic fibrosis (CF). However, the effect of inflammatory products on the repair capacity of respiratory epithelia is unclear. Our objective was to study the regulation of repair mechanisms by tumor necrosis factor-α (TNF-α), a major component of inflammation in CF, in a model of mechanical wounding, in two bronchial cell lines, non-CF NuLi and CF CuFi. We observed that TNF-α enhanced the NuLi and CuFi repair rates. Chronic exposure (24-48 h) to TNF-α augmented this stimulation as well as the migration rate during repair. The cellular mechanisms involved in this stimulation were then evaluated. First, we discerned that TNF-α induced metalloproteinase-9 release, epidermal growth factor (EGF) shedding, and subsequent EGF receptor transactivation. Second, TNF-α-induced stimulation of the NuLi and CuFi wound-closure rates was prevented by GM6001 (metalloproteinase inhibitor), EGF antibody (to titrate secreted EGF), and EGF receptor tyrosine kinase inhibitors. Furthermore, we recently reported a relationship between the EGF response and K(+) channel function, both controlling bronchial repair. We now show that TNF-α enhances KvLQT1 and K(ATP) currents, while their inhibition abolishes TNF-α-induced repair stimulation. These results indicate that the effect of TNF-α is mediated, at least in part, through EGF receptor transactivation and K(+) channel stimulation. In contrast, cell proliferation during repair was slowed by TNF-α, suggesting that TNF-α could exert contrasting actions on repair mechanisms of CF airway epithelia. Finally, the stimulatory effect of TNF-α on airway wound repair was confirmed on primary airway epithelial cells, from non-CF and CF patients.     

Radioreceptor assay for atrial natriuretic factor

Interest in accurate measurement of atrial natriuretic factor (ANF) in biological fluids and various tissues has been stimulated by recent data indicating the possible role of ANF in the homeostasis of salt and water. The presence of high-affinity binding sites for ANF in rat glomeruli has allowed us to develop a rapid, sensitive, and simple radioreceptor assay (RRA). A saturable high-affinity binding site on the membranes of rat glomeruli has been characterized by a dissociation constant of 33 pM and binding capacity of 396 fmol/mg protein. Rat plasma extracts or atrial homogenates or standards were incubated with radioiodinated ANF and a preparation of rat glomerular membranes. The receptor-bound and free radioactivity were separated by filtration on Whatman GF/C paper after 1 h incubation at room temperature. The sensitivity of the RRA was 2.08 fmol. The effective concentration of standard ANF that displaced 50% of labeled receptor-bound ANF (EC50) was 43.3 +/- 2.6 fmol/ml (n = 7). Both intra- and interassay coefficients of variation were smaller than 11%. This RRA assay has been compared with radioimmunoassay (RIA). High correlations for 19 plasma extracts and 34 atrial homogenates (r = 0.973 and r = 0.954, respectively) tested by RRA and RIA were obtained. This good correlation between the two methods suggests that the immunoreactive material found in rat plasma and atrial homogenates also displays biological activity.     

Evaluation of a radioreceptor assay to assess exogenous estrogen activity in serum of patients with breast cancer.

A radioreceptor assay (RRA) for the determination of total estrogen activity, was set up and used to assess the possible presence of exogenous molecules with estrogen activity in serum; a comparison was made with the specific radioimmunoassay (RIA) for the endogenous estrogen 17-B estradiol (17-B-E2). The assay was first performed on sera from healthy people taking estrogens in the form of oral contraceptives or lotions for local application whose total estrogenic activity in the blood was assumed to be abnormal. The assay was then performed on serum from 98 patients with early breast cancer and 20 patients with metastasis, not undergoing hormone therapy. A higher estrogen activity was found in 2.5% of sera compared to the activity found using the RIA method which is specific for endogenous estrogen 17-B-E2, the RRA/17-B-E2 ratio being higher than 3. Increased estrogen activity was found in 10% serum samples from digoxin treated cardiopathic patients, with an RRA/17-B-E2 ratio ranging from 4.4 to 20. The RRA assay could prove useful for showing up exogenous estrogen activity from various sources (drugs, food) in sera of people in whom estrogen stimulation could be potentially dangerous (i.e. in patients with hormone-sensitive tumors). This exogenous activity could support a certain degree of neoplastic stimulation and, therefore, unfavourably condition the patients' therapeutic response.     

Genotype-phenotype correlation in cystic fibrosis patients bearing [H939R;H949L] allele

Cystic fibrosis (CF) is caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations. We ascertained five patients with a novel complex CFTR allele, with two mutations, H939R and H949L, inherited in cis in the same exon of CFTR gene, and one different mutation per patient inherited in trans in a wide population of 289 Caucasian CF subjects from South Italy. The genotype-phenotype relationship in patients bearing this complex allele was investigated. The two associated mutations were related to classical severe CF phenotypes.