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PubMed1996

Modulation of epidermal differentiation, tissue inflammation, and T-lymphocyte infilitration in psoriatic plaques by topical calcitriol.

Psoriasis is characterized by immune activation, increased proliferation and abnormal differentiation of keratinocytes. The reported anti-psoriatic mechanisms of action in vivo of vitamin D analogues include reduction of keratinocyte proliferation and induction of keratinocyte terminal differentiation. We investigated whether the anti-psoriatic effect of the natural active vatamin D analogue, calcitriol, applied topically, is due to direct effects on keratinocytes alone or also due to immunoregulatory effects of calcitriol. Psoriasis patients were treated with topical calcitriol (0.005%) and a vehicle control for 8 weeks. Disease activity was assessed by a severity index and quantitative histopathological markers. In vitro studies of lymphocyte proliferation and gamma interferon secretion and of keratinocyte proliferation complemented the clinicohistopathologic studies. A heterogeneous response to calcitriol treatment could be segregated based upon elimination of K-16 keratin expression. Calcitriol treatment decreased keratinocyte proliferation, normalized keratinocyte differentiation and decreased immune activation in plaques. The histologic response to vitamin D treatment of psoriasis includes suppression of both immune and keratinocyte activation in situ. These studies provide a basis for rational combination of anti-psoriatic treatments and for the design of new vitamin D analogues to treat psoriasis.

Administration, Topical↗
PubMed2004

High throughput gene expression profiling: a molecular approach to integrative physiology.

Integrative physiology emphasizes the importance of understanding multiple pathways with overlapping, complementary, or opposing effects and their interactions in the context of intact organisms. The DNA microarray technology, the most commonly used method for high-throughput gene expression profiling, has been touted as an integrative tool that provides insights into regulatory pathways. However, the physiology community has been slow in acceptance of these techniques because of early failure in generating useful data and the lack of a cohesive theoretical framework in which experiments can be analysed. With recent advances in both technology and analysis, we propose a concept of multidimensional integration of physiology that incorporates data generated by DNA microarray and other functional, genomic, and proteomic approaches to achieve a truly integrative understanding of physiology. Analysis of several studies performed in simpler organisms or in mammalian model animals supports the feasibility of such multidimensional integration and demonstrates the power of DNA microarray as an indispensable molecular tool for such integration. Evaluation of DNA microarray techniques indicates that these techniques, despite limitations, have advanced to a point where the question-driven profiling research has become a feasible complement to the conventional, hypothesis-driven research. With a keen sense of homeostasis, global regulation, and quantitative analysis, integrative physiologists are uniquely positioned to apply these techniques to enhance the understanding of complex physiological functions.

Animals↗
PubMed1982

Minor coat protein composition and location of the A protein in bacteriophage f1 spheroids and I-forms.

The filamentous bacteriophage f1 can be transformed into a spherical particle (spheroid) or an intermediate shortened filament with a flared end (I-forms) by exposure to a chloroform-water interface at 22 or 4 degrees C, respectively. The protein composition of bacteriophage f1 spheroids and I-forms was examined by separating the proteins from the purified. [35S]cysteine-labeled particles by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. Quantitation of the radioactivity on the gels showed that I-forms and spheroids contain the same complement of minor coat proteins as do untreated f1 phage. This composition is unchanged after removal of the DNA, either by digestion with micrococcal nuclease or by centrifugation of the particles through CsCl density gradients, indicating that none of the minor coat proteins is held in the particles solely through an interaction with the DNA. We also examined the location of the A protein in I-forms by decoration with ferritin-conjugated antibodies and examination under the electron microscope and found that the A protein is located specifically at the flared end of the I-form particle, through which the DNA is extruded and at which contraction into spheroids begins. The implications of these results with regard to the orientation of the DNA within the capsid and the process of infection are discussed.

Capsid↗
PubMed1998

Increased immunoglobulin G production by short term cultured duodenal biopsy samples from HIV infected patients.

BACKGROUND: Secretory immunity is a major defence mechanism against infections at mucosal surfaces which are common in HIV infected patients. AIMS: To analyse intestinal immunoglobulin production in HIV infection in comparison with that in saliva and serum. PATIENTS AND METHODS: Immunoglobulin G (IgG), A (IgA), and M (IgM) concentrations were determined in supernatants of short term cultured duodenal biopsy samples, serum, and saliva from HIV infected patients (n = 28) and controls (n = 14) by radial immunodiffusion. RESULTS: IgG was increased in the supernatants of short term cultured biopsy samples and saliva from HIV infected patients compared with controls (p < 0.01), but IgA and IgM levels were normal. In contrast, both IgG and IgA concentrations in serum were higher in HIV infected patients than in controls (p < 0.002). No correlation was found between IgA produced by duodenal biopsy specimens and serum IgA. CONCLUSION: Abnormalities in mucosal immunoglobulin production in HIV infection were surprisingly small, indicating that specific secretory immunity rather than quantitative immunoglobulin production may be impaired. However, increased production of IgG could contribute to mucosal inflammation by complement activation. Our findings of normal mucosal IgA production and the lack of correlation between serum and mucosal IgA argues against an intestinal origin for the increased serum IgA levels in HIV infected patients.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Impact of the phytoestrogen content of laboratory animal feed on the gene expression profile of the reproductive system in the immature female rat.

The effect of the dietary background of phytoestrogens on the outcome of rodent bioassays used to identify and assess the reproductive hazard of endocrine-disrupting chemicals is controversial. Phytoestrogens, including genistein, daidzein, and coumestrol, are fairly abundant in soybeans and alfalfa, common ingredients of laboratory animal diets. These compounds are weak agonists for the estrogen receptor (ER) and, when administered at sufficient doses, elicit an estrogenic response in vivo. In this study, we assessed the potential estrogenic effects of dietary phytoestrogens at the gene expression level, together with traditional biologic end points, using estrogen-responsive tissues of the immature female rat. We compared the gene expression profile of the uterus and ovaries, as a pool, obtained using a uterotrophic assay protocol, from intact prepubertal rats fed a casein-based diet (free from soy and alfalfa) or a regular rodent diet (Purina 5001) containing soy and alfalfa. Estrogenic potency of the phytoestrogen-containing diet was determined by analyzing uterine wet weight gain, luminal epithelial cell height, and gene expression profile in the uterus and ovaries. These were compared with the same parameters evaluated in animals exposed to a low dose of a potent ER agonist [0.1 microg/kg/day 17alpha-ethynyl estradiol (EE) for 4 days]. Exposure to dietary phytoestrogens or to a low dose of EE did not advance vaginal opening, increase uterine wet weight, or increase luminal epithelial cell height in animals fed either diet. Although there are genes whose expression differs in animals fed the soy/alfalfa-based diet versus the casein diet, those genes are not associated with estrogenic stimulation. The expression of genes well known to be estrogen regulated, such as progesterone receptor, intestinal calcium-binding protein, and complement component 3, is not affected by consumption of the soy/alfalfa-based diet when assessed by microarray or quantitative reverse transcriptase-polymerase chain reaction analysis. Our results indicate that although diet composition has an impact on gene expression in uterus and ovaries, it does not contribute to the effects of an ER agonist.

Animal Feed↗
PubMedDate not supplied

Real-time polymerase chain reaction: a novel molecular diagnostic tool for equine infectious diseases.

The focus of rapid diagnosis of infectious disease of horses in the last decade has shifted from the conventional laboratory techniques of antigen detection, microscopy, and culture to molecular diagnosis of infectious agents. Equine practitioners must be able to interpret the use, limitations, and results of molecular diagnostic techniques, as they are increasingly integrated into routine microbiology laboratory protocols. Polymerase chain reaction (PCR) is the best-known and most successfully implemented diagnostic molecular technology to date. It can detect slow-growing, difficult-to-cultivate, or uncultivatable microorganisms and can be used in situations in which clinical microbiology diagnostic procedures are inadequate, time-consuming, difficult, expensive, or hazardous to laboratory staff. Inherent technical limitations of PCR are present, but they are reduced in laboratories that use standardized protocols, conduct rigid validation protocols, and adhere to appropriate quality-control procedures. Improvements in PCR, especially probe-based real-time PCR, have broadened its diagnostic capabilities in clinical infectious diseases to complement and even surpass traditional methods in some situations. Furthermore, real-time PCR is capable of quantitation, allowing discrimination of clinically relevant infections characterized by pathogen replication and high pathogen loads from chronic latent infections. Automation of all components of PCR is now possible, which will decrease the risk of generating false-positive results due to contamination. The novel real-time PCR strategy and clinical applications in equine infectious diseases will be the subject of this review.

Animals↗
PubMed1988

Relationship between membrane depolarization and extracellular calcium influx during neutrophil activation.

To better define the relationship between membrane depolarization and extracellular Ca2+ influx during neutrophil activation, we compared stimulation by elevating the extracellular K+ concentration, [K+]o, with stimulation by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP). Elevation of [K+]o resulted in uniform depolarization of the entire population of cells. This was associated with an influx of Ca2+ that was temporally delayed and quantitatively less than that induced by fMLP. K+ depolarization also caused increased expression of type 1 (C3b/C4b) complement receptor (CR1) and type 3 (C3bi) complement receptor (CR3), but the increments were less than with fMLP. We then used pertussis toxin to determine if guanosine triphosphate (GTP)-binding proteins were involved in these responses. Toxin inhibited the fMLP-induced membrane depolarization as well as the uptake of extracellular Ca2+ and the expression of both CR1 and CR3 induced by the chemoattractant. This indicates that the fMLP receptor is not directly coupled to an ion channel. The membrane depolarization induced by elevating [K+]o was not inhibited by toxin, but the uptake of Ca2+ and the increased expression of CR1 and CR3 were all significantly inhibited. The toxin failed to block increased CR1 and CR3 expression induced by ionomycin, demonstrating that its effects were not attributable to general toxicity. The results suggest that voltage gating is not the major mechanism by which polymorphonuclear leukocytes (PMNs) increase their permeability to extracellular Ca2+. Initial signals, whether generated by chemoattractants binding to their receptors or by small initial influxes of extracellular Ca2+, must be amplified by pertussis toxin-sensitive steps to fully increase the Ca2+ permeability and optimally activate the cell.

Calcium↗
PubMed1983

C1 inactivator-C1s complexes in inflammatory joint disease.

A newly developed enzyme linked immunosorbent assay for the quantitation of C1 inhibitor (C1In)-C1s complexes was used to study activation of the classical pathway of complement in inflammatory joint diseases. Synovial fluid (SF) specimens were obtained from patients with rheumatoid arthritis (RA), other arthritides and non-inflammatory joint effusions. Paired serum (S) samples were obtained in 17 cases. Immune complexes (IC) were measured by the staphylococcal binding assay. C1In-C1s were higher in RA SF samples than in paired RAS samples (P less than 0.01). IC were higher in RA SF than non-RA SF. There was a significant inverse correlation between SF C1In-C1s complexes and SF total haemolytic complement. For all SF samples there was a correlation between IC and C1In-C1s complexes, but for RA SF alone there was no significant correlation between these parameters. There was no correlation between titre of rheumatoid factor and C1In-C1s complexes. These results demonstrate that activation of the classical pathway of complement is the hallmark of rheumatoid synovitis, yet also suggest functional heterogeneity of both circulating and intra-articular IC.

Antigen-Antibody Complex↗
PubMed1981

Genetic linkage of resistance to Listeria monocytogenes with macrophage inflammatory responses.

The mobilization of adequate numbers of mononuclear phagocytes to inflammatory foci was measured in Listeria-resistant and Listeria-sensitive mice. Resistant strains, such as B10.A, were found to have a 2- to 3-fold greater accumulation of peritoneal macrophages after i.p. treatment with a variety of nonspecific inflammatory stimuli in comparison to sensitive strains, such s A/J. In addition to low macrophage inflammatory responses, A/J mice had fewer resident peritoneal macrophages. Moreover, measurement of chemotaxis in vitro of equal numbers of thioglycollate-induced macrophages showed cells from A/J mice to be less responsive to complement-derived chemotactic factors. Thus, the mononuclear phagocyte systems of resistant B10.A and sensitive A/J mice were quantitatively and qualitatively different. A survey of inbred mouse strains revealed that these differences were not peculiar to A strain mice and that, in general, the level of the in vivo macrophage inflammatory response correlated with the level of resistance to Listeria in a given strain. Like resistance to Listeria, the macrophage inflammatory response was found to be genetically controlled by an autosomal, non-H-2-linked gene(s) expressed as incompletely dominant. Backcross analysis showed genetic linkage of the macrophage inflammatory response with resistance to Listeria. Thus, the results of this study provide formal evidence that the cellular basis of the genetically determined, enhanced resistance to Listeria is an augmented pool size of mononuclear phagocytes, and the prompt mobilization of these cells to foci of infection.

Animals↗
PubMed1986

Complement activation in vitro by antiplatelet antibodies in chronic immune thrombocytopenic purpura.

Many chronic ITP patients have increased amounts of platelet-associated IgG, C3, C4 and C9, suggesting in vivo complement activation. In this study, we assessed the ability of various antiplatelet antibodies (APA) to activate and deposit complement proteins and to cause platelet lysis in vitro. Platelet sensitization with rabbit APA, anti-P1A1 antibody (one patient), anti-HLA antibody (two patients) and ITP autoantibodies (four patients) resulted in the deposition of C4 and C3 onto platelets in an amount proportional to the quantity of antibody-containing sera used to sensitize the platelets. Although C9 deposition onto platelets could not be quantitatively demonstrated on platelets sensitized with ITP serum, platelet lysis (51Cr release) was noted after incubation with each of three ITP sera and complement. When compared, ITP autoantibodies, anti-HLA and anti-P1A1 antibodies activated complement to a similar degree. We conclude that some autoantibodies in chronic ITP activate the classical complement pathway. The demonstration of in vitro platelet lysis by autoantibodies and complement suggests that in vivo platelet lysis may occur in some chronic ITP patients.

Autoantibodies↗
PubMed1987

Activation of factor B of the alternative pathway of complement. Assessment by rocket immunoelectrophoresis.

During activation of the alternative pathway of complement, Factor B is cleaved into a smaller fragment Ba and a larger fragment Bb. The Ba in plasma was quantitated by one-dimensional rocket immunoelectrophoresis after precipitating plasma B and Bb with 21% polyethyleneglycol. This method was much more sensitive and faster than other technics with which it was compared. Fragment Ba was detected in 22 of 28 rheumatoid arthritis plasma samples and in 12 of 15 systemic lupus erythematous samples but in only 8 of 68 samples obtained from healthy volunteers. The traditional approach of measuring total Factor B for the assessment of activation of the alternative pathway showed values that were even higher than normal in these diseases. The measurement of plasma Ba promises to be a valuable means of assessment of the activation of Factor B and the alternative pathway of complement in clinical disorders.

Arthritis, Rheumatoid↗
PubMed1993

The role of I and B in peritonitis associated with the nephrotic syndrome of childhood.

Children with nephrotic syndrome (NS) are susceptible to bacterial infections, including primary bacterial peritonitis. Immunologic abnormalities associated with NS include low serum levels of the complement proteins I and B of the alternative complement pathway. We developed a novel and highly sensitive enzyme immunoassay using murine MAb to I and B to quantitate urinary concentrations of these proteins. We studied 22 patients with minimal change NS, including seven with a history of peritonitis (1.6 +/- 0.3 episodes, mean +/- SEM) and 15 without such a history. The two groups did not differ significantly in age, sex, race, age at onset of disease, or duration of disease. Children with minimal change NS complicated by peritonitis had 1) increased urinary excretion of both I (p < 0.05) and B (p < 0.05) in relapse versus remission, 2) greater excretion of I in both relapse (p < 0.01) and remission (p < 0.05) compared with patients without peritonitis, 3) greater excretion of B in relapse compared with patients without peritonitis (p < 0.05), and 4) decreased plasma levels of I compared with patients without peritonitis and controls (p < 0.01) and decreased plasma levels of B compared with controls. Increased urinary excretion of I correlated with decreased plasma levels of I (r = 0.88, p < 0.001). These data support our initial hypothesis that depressed plasma concentrations of these proteins of the alternative complement pathway may predispose patients with minimal change NS to peritonitis and that urinary loss of these proteins is a tenable mechanism.

Child↗
PubMed1987

Activation of terminal components of complement in patients with Guillain-Barré syndrome and other demyelinating neuropathies.

In the present study, the role of antiperipheral nerve myelin antibody (anti-PNM Ab) in demyelination by generating the terminal attack complex (C5b-9) of complement was explored in patients with Guillain-Barré syndrome (GBS) and other demyelinating neuropathies. The presence in serum of SC5b-9, an inactive C5b-9 containing S protein, was assessed quantitatively by enzyme-linked immunosorbent assay using an antibody (Ab) to neoantigens expressed on C9 when complexed with C5b-8 or after tubular polymerization. SC5b-9 was detected in all 19 GBS, four patients with paraprotein-associated neuropathy and five of six patients with chronic recurrent polyneuritis. No SC5b-9 was detected in 10 normal controls. Kinetic studies from six GBS patients showed the highest values of SC5b-9 on the 3rd to 5th d of admission; in contrast, the anti-PNM Ab were highest on the day of admission. Anti-PNM Ab fell rapidly to very low levels by the 15th to 20th d. SC5b-9 declined with similar kinetics to undetectable levels by the 30th d. Levels of Ab and SC5b-9 did not quantitatively correlate with soluble immune complexes in these patients' serum. Membrane-bound C5b-9 was also detected by immunohistochemistry in the peripheral nerves from a GBS patient. These results, which show a relationship between levels of complement-fixing anti-PNM Ab and the tissue-damaging C5b-9 complex, suggest that peripheral nerve myelin may serve as the target for Ab-mediated complement attack.

Antigen-Antibody Complex↗
PubMed1980

Partial plasma exchange using albumin replacement: removal and recovery of normal plasma constituents.

Using albumin and crystalloid as the only replacement fluids, the effect of partial plasma exchange on the removal and recovery of normal plasma constituents was studied. The results of 30 procedures on 10 individuals were evaluated. Four patterns of removal are described: reduction in the concentration of fibrinogen and C3 were greater than would be expected based upon the extent of the exchange, while IgG, IgM, cholesterol, alkaline phosphatase and SGPT were removed as expected. Reduction of serum glutamicoxalacetic transaminase (SGOT), lactate dehydrogenase (LDH), amylase, and creatine phosphokinase (CPK) averaged 17% less, and uric acid, calcium and K+ averaged 53% less than expected. Concentrations of HCO-3 and glucose did not change. The mean recovery for all constituents except fibrinogen, C3, cholesterol. IgG and IgM was near 100% at 48-72 hr postpheresis. The 72-hr recovery of fibrinogen and complement was 66% and 60%, respectively. Cholesterol recovery was also slow, requiring a minimum of 1 wk to reach prepheresis levels. Measured at a time when quantitative IgM levels were still reduced, alloantibody agglutinating activity (anti-A and anti-B) in a postpheresis sample exceeded prepheresis agglutinating activity. These data demonstrated that, depending upon quantity and frequency of pheresis, partial plasma exchange using albumin replacement may cause progressive marked reduction in concentrations of immunoglobulin, complement, fibrinogen, and cholesterol. Furthermore, newly synthesized antibody may have increased biologic activity.

Alanine Transaminase↗
PubMed1995

Quantitative differences in adhesiveness of type 1 fimbriated Escherichia coli due to structural differences in fimH genes.

Type 1 fimbriae are heteropolymeric surface organelles responsible for the D-mannose-sensitive (MS) adhesion of Escherichia coli. We recently reported that variation of receptor specificity of type 1 fimbriae can result solely from minor alterations in the structure of the gene for the FimH adhesin subunit. To further study the relationship between allelic variation of the fimH gene and adhesive properties of type 1 fimbriae, the fimH genes from five additional strains were cloned and used to complement the FimH deletion in E. coli KB18. When the parental and recombinant strains were tested for adhesion to immobilized mannan, a wide quantitative range in the ability of bacteria to adhere was noted. The differences in adhesion do not appear to be due to differences in the levels of fimbriation or relative levels of incorporation of FimH, because these parameters were similar in low-adhesion and high-adhesion strains. The nucleotide sequence for each of the fimH genes was determined. Analysis of deduced FimH sequences allowed identification of two sequence homology groups, based on the presence of Asn-70 and Ser-78 or Ser-70 and Asn-78 residues. The consensus sequences for each group conferred very low adhesion activity, and this low-adhesion phenotype predominated among a group of 43 fecal isolates. Strains isolated from a different host niche, the urinary tract, expressed type 1 fimbriae that conferred an increased level of adhesion. The results presented here strongly suggest that the quantitative variations in MS adhesion are due primarily to structural differences in the FimH adhesin. The observed differences in MS adhesion among populations of E. coli isolated from different host niches call attention to the possibility that phenotypic variants of FimH may play a functional role in populations dynamics.

Adhesins, Bacterial↗
PubMed1990

Synthesis of complement component C5 by human B and T lymphoblastoid cell lines.

Human B and T lymphoblastoid cell lines were shown to synthesize C5. C5 synthesis was quantitated with an enzyme-linked immunosorbent assay (ELISA) that utilized a pool of C5-specific monoclonal antibodies (mAbs). Some level of C5 synthesis was detected in all eight of the B and T cell lines examined. In three of the cell lines, C5 was detected in both culture supernatants and whole cell detergent lysates, whereas in the other five cell lines, C5 was detected only in the cell lysates. Lymphoblastoid cells with both distributions of C5 were shown to synthesize a messenger RNA that was similar in size to the C5 mRNA expressed by the HepG2 hepatoma cell line. Estimates of the concentration of the C5 transcript in poly(A)+ RNA from lymphoblastoid and HepG2 cells suggested that C5 mRNA levels in the lymphoblastoid cell lines were comparable and about one-tenth of the levels in HepG2 cells. Lymphoblastoid C5, isolated by immunoaffinity chromatography from the supernatants of 35S-labeled cultures, had the same subunit composition as plasma-derived C5, but had an alpha subunit of slightly smaller relative mass.

B-Lymphocytes↗
PubMed1988

Macromolecular organization of natural and recombinant lung surfactant protein SP 28-36. Structural homology with the complement factor C1q.

The macromolecular structure of the pulmonary surfactant apolipoprotein SP 28-36 has been determined. For SP 28-36 isolated from dog lung lavage, a flower bouquet-like hexameric structure with six globular domains connected by short stalks to a common stem was revealed by electron microscopy, using the rotary shadowing technique. This structure is very similar to that published for the subcomponent C1q of the first component of complement C1. The lavage material was compared with the homologous human recombinant SP 28-36 by the same technique. Mostly smaller aggregates like di-, tri- and tetramers as well as very high aggregates were observed. Mild reduction of the recombinant material revealed the lollipop-shaped monomers composed of a globular domain and a tail with a discrete kink in the middle portion. The collagenous nature of the tail was demonstrated by circular dichroism spectroscopy. This implies that the mammalian expression system assembles the monomeric subunits correctly. Assembly into the hexameric structures, however, does not proceed quantitatively.

Animals↗
PubMed1983

Complement activation in shock associated with a surgically provoked bacteriaemia.

The activation of complement (C) factors in a patient exhibiting an anaphylactoid shock 10 min after the initiation of transurethral coagulation of vesical papillomas was studied. Streptococcus faecalis was cultured from he urine before and after the shock and from the blood on two occasions within 2 days after the shock. The shock was associated with a marked but transient complement activation as judged from serial determinations of C3d and C3c by doubledecker rocket immuno electrophoresis and native C3 and C3c by crossed immuno electrophoresis. Conventional total C3 quantitation by rocket immuno electrophoresis did not reveal the activation. A decrease of native C4 to 20% of normal values and the appearance of C4 split products indicated classical pathway activation. Factor B conversion showed the alternative pathway to be involved as well. Total haemolytic complement activity was temporarily reduced, increasing without reaching normal values within 20 hours. The results indicated that the classical pathway was transiently activated, pointing to an acute antigen-antibody reaction, probably caused by the entrance of bacteria and bacterial products into the circulation of an immunized host.

Anaphylaxis↗