Humor Fluid Research Articles

Identification and expression of a ferritin homolog in amphioxus Branchiostoma belcheri: Evidence for its dual role in immune response and iron metabolism

Ferritin plays a key role in cellular iron metabolism including iron storage and detoxification, which has been identified in a wide range of organisms including bacteria, fungi, plants and animals. However, little information is available regarding ferritin in the protochordates to date. Here we demonstrate the presence of a ferritin gene homolog, BbFRT, in amphioxus Branchiostoma belcheri. Analysis of the BbFRT 5′-UTR indicated the existence of a putative iron-responsive element (IRE) with a predicated stem-loop structure. BbFRT encoded a deduced protein of 172 amino acids with the conserved motif for ferroxidase center typical of heavy chains of vertebrate ferritins. Sequence comparison showed that BbFRT shared more identity to H-chains (68%) of vertebrate ferritins than to the L-chains (46–51%). Both in situ hybridization histochemistry and immunohistochemical staining revealed that BbFRT was ubiquitously expressed in B. belcheri. In addition, BbFRT expression was up-regulated by 1.6-fold and 1.5-fold, respectively, following exposure to LPS at both transcriptional and translational levels. Similarly, exposure to iron resulted in about 1.6-fold increase in BbFRT in the humoral fluids. These suggest that BbFRT seems a protein with a dual function functioning in both immune response and iron metabolism.

Pathophysiological Features of Endogenous Intoxication in Pregnant Women with Arterial Hypertension

Objective: to determine the nature and specific features of development of endogenous intoxication in pregnant women with arterial hypertension. Subjects and materials. Humoral extracellular fluid volume regulation, partial renal functions, placental hormonal function, membranous lipid peroxidation activity, antiradical defense, the parameters of central hemodynamics, endogenous intoxication, and a biochemical coagulogram were studied and differential blood count with the leukocytic ratio indices was estimated in 172 pregnant females with arterial hypertension and 54 healthy pregnant ones in the third trimester. The statistical package «Stadia» was applied. Results. Arterial hypertension caused by pregnancy was ascertained to involve pathogenetically different types: low-, normal-, and high-renin ones. In pregnant women with arterial hypertension, the general pathogenetic homeostatic changes were placental hormonal imbalance, activated membranous lipid peroxidation, impaired lymph outflow, sodium and water retention, hepatic and renal failure, and endogenous intoxication. Conclusion. Placental ischemia appearing as placental hormonal imbalance (extrarenal pressor system) was accompanied by a compensatory humoral response: arterial hypertension and metabolic disturbances. Changes in medium-weight molecule 280, leukocytic intoxication index, erythrocytic sorption capacity, and Paramecium test, by confirming the presence of endogenous intoxication in pregnant females with arterial hypertension, were caused by a type of arterial hypertension (by the hemodynamic profile and the type of impaired partial renal functions). Key words: pregnancy, arterial hypertension, endogenous intoxication.

Ficolins: Novel pattern recognition molecules of the innate immune response

Ficolins are members of the collectin family of proteins which are able to recognize pathogen-associated molecular pattern (PAMP) on microbial surfaces. Upon binding to their specific PAMP, ficolins may trigger activation of the immune system by either binding to cellular receptors for collectins or by initiating activation of complement via the lectin pathway. For the latter, the human ficolins (i.e. L-, H- and M-ficolin) and murine ficolin-A were shown to associate with the lectin pathway-specific serine protease MBL-associated serine protease-2 (MASP-2) and catalyse its activation which in turn activates C4 and C4b-bound C2 to generate the C3 convertase C4b2a. There is mounting evidence underlining the lectin nature of ficolins with a wide range of carbohydrate moieties recognized on microbial surfaces. However, not all members of the ficolin family appear to act as lectin pathway recognition components. For example, murine ficolin-B does not associate with MASP-2 and appears to be absent in plasma and other humoral fluids. Its stringent cellular localization points to other functions within the immune response, possibly acting as an intracellular scavenger to target and facilitate clearance of PAMP-bearing debris. When comparing ficolin orthologues from different species, it appears evident that human, murine, and porcine ficolins differ in many aspects, a specific point that we aim to address in this review.

Complement-mediated killing of Vibrio species by the humoral fluids of amphioxus Branchiostoma belcheri: Implications for a dual role of O-antigens in the resistance to bactericidal activity

The functional properties of complement in invertebrate deuterostomes are rather ill-defined. Here we showed that the humoral fluids from amphioxus Branchiostoma belcheri were capable of causing lysis of some Vibrio species including Vibrio alginolyticus HW284, Vibrio parahaemolyticus HW458 and Vibrio harvey SF-1, the first such data in the invertebrate deuterostomes. The fluid bacteriolytic activity was abolished by pre-incubation with heat-inactivated rabbit anti-human C3 serum, heating at 45 °C for 30 min, and repeated thawing and freezing. Additionally, the bacteriolytic activity was Mg 2+-dependent and Ca 2+-independent, and selective activation of the alternative pathway by zymosan A induced a loss of bacteriolytic activity. This strongly suggests that activation of the alternative complement pathway is responsible for the fluid bacteriolytic activity. It was also shown that some Vibrio species like Vibrio cincinnatiensis HW287 appeared resistant to the complement-mediated lysis. The LPS profiling revealed that the fluid-resistant V. cincinnatiensis HW287 had an LPS profile with a ladder of both high-molecular-weight (HMW) and low-molecular-weight (LMW) O-antigen bands, whereas the fluid-sensitive V. alginolyticus HW284 had few HMW O-antigen bands, suggesting a positive correlation between O-antigen size and humoral fluid resistance. Moreover, complement consumption assays demonstrated that both V. alginolyticus HW284 and V. cincinnatiensis HW287 consumed complement, with the former consuming significantly higher complement than the latter. Overall, it is suggested that HMW O-antigens may protect the fluid-resistant Vibrio species by a dual act of avoiding initiating complement activation as well as sterically hindering complement from gaining access to and damaging the cell membrane.

Proteomic Approach for Caudal Trauma-Induced Acute Phase Proteins Reveals That Creatine Kinase Is a Key Acute Phase Protein in Amphioxus Humoral Fluid

Elevated creatine kinase (CK) in the circulation was generally regarded to be a passive release from muscle damage. We utilized proteomic methodologies to characterize amphioxus humoral fluid APPs in response to caudal trauma, and found several spots of CK alterations with up-regulation and pI shift. Its amount and enzyme activity showed a dynamic pattern of APP in humoral fluid accompanied with a reduction in enzyme activity of muscle, whereas there was no significant difference in CK amount of muscle and the other tissues and in CK enzyme activity of the other tissues between different time points of sample collection following caudal trauma. In addition, CK phosphorylation regulation during injury was not achieved by monoclonal antibodies separately against phosphothreonine, phosphotyrosine, and phosphoserine. These results suggested that the CK elevation of humoral fluid might be from muscle, being an active response to caudal trauma rather than a passive release from muscle damage. Therefore, CK ability in response to caudal trauma should be highly concerned.

Assessing Free and Total Morphine following Heroin Overdose when Complicated by the Presence of Toxic Amitriptyline Levels

A 43-year-old female was reported to inject heroin, which led to her rapid death. Because of the potential for criminal charges, laboratory results that could verify "hotshot" heroin overdose were valuable. Initial toxicological analysis detected morphine (0.78 mg/L), amitriptyline (2.91 mg/L), and nortriptyline (2.80 mg/L) in femoral blood. Because these tricyclic antidepressant levels alone might normally be associated with a fatal outcome, the ratio of free versus total morphine (88.6%) and presence of 6-monoacetylmorphine in vitreous fluid were used support a history of rapid death following intravenous (IV) administration. The distribution of amitriptyline and nortriptyline was consistent with accumulation of drug after chronic dosing. Our other results suggest that the low morphine level in vitreous humor fluid (0.16 mg/L) relative to free morphine in femoral blood (0.78 mg/L) may also be an indicator of limited survival time following exposure to morphine. Based upon comprehensive toxicologic analysis, we determined overdose due to IV abuse of heroin was likely to have precipitated the fatal outcome. This case underscores the need for complete toxicologic workup and to consider individual variation in the dose response during toxicologic interpretation of postmortem results.

Purification and characterization of lectin from humoral fluids ofCharybdis feriatus

To search new sources of lectin, an experiment on lectin distribution in humoral serum of crabCharybdis feriatus (in short, CFL) was conducted March, 2002. When adding solid ammonium sulfate into the fluids up to 50% saturation at 4°C, most CFL activity showed precipitates who were then continually extracted by ammonium sulfate of different concentrations. The supernatant, which was called primary CFL fluids, was given a 17.60-fold purification and 45.70% recovery of total activity. Finally, by using Sephadex G-100 column chromatography, the CFL in the primary CFL fluid was highly purified. Compared to the original humoral fluids, the last purified CFL got a 203.90-fold purification and 30.48% recovery of total activity, and demonstrated a single band on SDS-PAGE. In the same time, the purified CFL was detected for agglutination activity with 7 kinds of animal erythrocytes. Other characterization, such as sugar inhibition, and the effect of temperature on the agglutination activity of CFL were also studied. Our results indicate that agglutination activity of CFL was influenced by sugar and temperature.

Alanine Aminotransferase in Amphioxus: Presence, Localization and Up-regulation after Acute Lipopolysaccharide Exposure

Alanine aminotransferase (AAT) is mainly synthesized in the liver, and its level in mammalian serum is elevated after acute phase induction. Here we demonstrated that sheep anti-human AAT antibody cross-reacted with amphioxus humoral fluids as well as human serum; and the concentration of AAT in the humoral fluids in amphioxus increased after the acute challenge with lipopolysaccharide, while the level of total proteins remains unchanged. These suggest the presence of the same acute phase response pattern in amphioxus, as observed in some mammalian species. Immunohistochemically, AAT was localized in the hepatic diverticulum, ovary and testis. It appears that the hepatic diverticulum in amphioxus is functionally homologous to the vertebrate liver in respect of AAT synthesis, supporting the hypothesis that the vertebrate liver evolved from the hepatic diverticulum of an amphioxus-like ancestor during early chordate evolution.

Immunohistochemical localization of vitellogenin in the hepatic diverticulum of the amphioxus Branchiostoma belcheri tsingtauense, with implications for the origin of the liver

Abstract. The localization of vitellogenin (Vg) remains untested in amphioxus. Western blotting analysis showed that mouse anti‐amphioxus Vg sera cross‐reacted with the crude extracts of the hepatic diverticulum and ovary, and humoral fluids, including blood and coelomic fluid, from specimens of Branchiostoma belcheri. Similarly, immunohistochemical staining also revealed that Vg is localized in the hepatic diverticulum, sub‐intestinal vessels, and ovary in amphioxus. The hepatic diverticulum of amphioxus, like the vertebrate liver, synthesizes Vg, suggesting that amphioxus hepatic diverticulum is functionally equivalent to the vertebrate liver in respect of the synthesis of this compound. This agrees with the idea that amphioxus hepatic diverticulum is the precursor of vertebrate liver.

Demonstration of plasminogen‐like protein in amphioxus with implications for the origin of vertebrate liver

AbstractPlasminogen, the proenzyme of serine protease plasmin, is a plasma glycoprotein synthesized primarily in the liver, and its evolutionary origin in chordates remains unclear. We demonstrated here that the humoral fluid in amphioxus is capable of cross‐reacting with anti‐human or anti‐mouse plasminogen antibodies, and the hepatic diverticulum in amphioxus is the site of plasminogen‐like protein synthesis. The presence of plasminogen‐like protein in amphioxus pushes the origin of plasminogen to before the last common ancestor of vertebrates. In addition, the localization of plasminogen‐like protein in the hepatic diverticulum suggests that the diverticulum in amphioxus is functionally homologous to the vertebrate liver in respect of plasminogen synthesis, supporting the hypothesis that the vertebrate liver evolved from the hepatic diverticulum of an amphioxus‐like ancestor during early chordate evolution.

Humoral immune responses of amphioxus Branchiostoma belcheri to challenge with Escherichia coli

Humoral parameters of amphioxus Branchiostoma belcheri, including lysozyme, antimicrobial activity, microbial agglutinin and haemagglutinins were measured before and after challenge with Escherichia coli. Humoral fluids from unchallenged B. belcheri had lysozyme, antimicrobial, microbial agglutinating and haemagglutinating activities, which may represent part of the baseline level of innate immunity in this organism. After challenge with E. coli, the lysozyme activity, growth-inhibiting activities against E. coli and Vibrio alginolyticus, microbial agglutinating activities against Micrococcus lysodeikticus, Bacillus subtilis and Staphylococus aureus, and haemagglutinating activities against rabbit and human A and O erythrocytes in the humoral fluids were all increased significantly. In contrast, the agglutinating activities against Vibrio harveyi and E. coli and the haemagglutinating activity against human B erythrocytes in the humoral fluids were reduced in response to E. coli challenge. It appears that the humoral fluids of B. belcheri contain components that are able to differentiate different microbes and different human blood cell types.

Presence and localization of antithrombin and its regulation after acute lipopolysaccharide exposure in amphioxus, with implications for the origin of vertebrate liver

Antithrombin (AT), which is mainly synthesized in the liver, is an acute-phase plasma protein in mammalian species. Here, we demonstrated that sheep anti-human AT antibody cross-reacted with the humoral fluids in amphioxus Branchiostoma belcheri tsingtauense as well as human serum. The concentration of AT in the humoral fluids in amphioxus decreased slightly at first and then increased after the acute challenge with lipopolysaccharide, while the level of total proteins remained unchanged. These suggest the presence of the same acute-phase response pattern in amphioxus, as observed in some mammalian species. Immunohistochemically, AT was localized in the hepatic diverticulum. It is clear that the hepatic diverticulum in amphioxus is homologous to the vertebrate liver with respect to AT synthesis. This lends support to the hypothesis originally suggested by Müller that the vertebrate liver evolved from the hepatic diverticulum of an amphioxus-like ancestor during early chordate evolution.

Purification and characterisation of phenoloxidase from amphioxus Branchiostoma belcheri tsingtauense

Phenoloxidase (PO) from the humoral fluid of amphioxus B. belcheri tsingtauense was purified using a sequential combination of ammonium sulphate precipitation, Sephadex G-200 chromatography and DEAE Sepharose™ Fast Flow chromatography. In PAGE, the purified enzyme exhibited a single band of 150 kDa under non-reducing conditions, and was resolved to three bands with molecular masses of 72, 46 and 44 kDa, respectively, under reducing conditions, suggesting that the PO in amphioxus humoral fluid seems to be a heterotrimer of three polypeptides held together by disulphide bonds. The substrate specificity and inhibition characteristics both indicate that the PO isolated from amphioxus humoral fluid is a tyrosinase-type enzyme. In addition, mouse antisera against the purified PO were prepared, and their specificity was confirmed by Western blotting, facilitating the future determination of the origin of PO in the humoral fluid and the distribution of PO-synthesising tissues in amphioxus.

Presence of prophenoloxidase in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense

The presence of phenoloxidase (PO) activity in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense was electrophoretically and spectrophotometrically studied. The enzyme was present in the humoral fluid predominantly as an inactive proenzyme, prophenoloxidase (proPO). The optimum temperature for activation of the proPO ranged from 30 °C to 35 °C, and the enzyme exhibited optimum activity at pH between 7.0 and 7.5. ProPO in the humoral fluid was readily activated to active form PO by exogenous elicitors such as trypsin, zymosan and LPS. The activation of the proPO by exogenous elicitors was significantly enhanced in the presence of 10 mM Ca 2+, but was susceptible to serine protease inhibitors like soybean trypsin inhibitor and p-nitrophenyl- p′-guanidinobenzoate. PAGE revealed a single band of PO activity in the humoral fluid with an apparent molecular mass of 150 kDa, which was resolved to three bands with molecular masses of 44, 46 and 72 kDa, respectively, after SDS-PAGE. This is the first report on the presence of the enzyme PO in amphioxus humoral fluid.

Bcl-2 expression by CD4 T lymphocytes in Vogt-Koyanagi-Harada disease

Purpose: To determine whether Bcl-2 is expressed on CD 4 + lymphocytes in the aqueous humor (AH) and cerebrospinal fluid (CSF) of patients with Vogt-Koyanagi-Harada (VKH) disease, and to determine whether Fas will induce apoptosis of lymphocytes in the CSF. Methods: The percentages of CD4, CD8, CD45RO, Fas, and Bcl-2 positive T lymphocytes in the AH and CSF of eight patients with active VKH and five healthy controls were determined by flow cytometry. Soluble Fas ligand (sFasL) in the CSF was measured by ELISA. Freshly isolated cells from the CSF were cultured with anti-Fas antibody (Ab) and apoptosis was assessed by the TUNEL method. Results: Fas + CD4 + lymphocytes were the predominant lymphocytes in the AH and CSF of VKH patients. Bcl-2 was strongly expressed in these cells. Soluble FasL was also detected in the CSF. The number of apoptotic cells detected by anti-Fas Ab was not significantly increased in the CSF of VKH patients. Conclusions: In spite of the high expression of Fas antigen on CD4 + cells and the presence of sFasL in the CSF, apoptosis was not observed. Bcl-2 expression may contribute to the regulation of apoptosis of inflammatory cells in the CSF of VKH patients.

Presence and induction by bacteria of D-galactoside-specific lectins in the humoral fluids of amphioxus Branchiostoma belcheri tsingtauense

The presence of lectins in the humoral fluids of amphioxus, an animal transitional from invertebrates to vertebrates, has thus far not been reported. It was shown here that lectins are present in the humoral fluids of amphioxus. The lectin activity in the humoral fluids of amphioxus is SH group-dependent and is specifically inhibited by various D-galactosides. Also, the humoral fluids obtained from E. coli-injected amphioxus showed increased agglutinating activity against human B and O, rabbit, grass carp and toad erythrocytes, suggesting that a form of adaptive response might exist in amphioxus.

Soluble Fas ligand expression in the ocular fluids of uveitis patients

Purpose. The expression of Fas ligand (FasL) in ocular tissues is thought to play a critical role in maintaining immune privilege in the eye. In this study, to clarify the involvement of the Fas-FasL system in inflammatory processes of the eye,we examined soluble FasL (sFasL) in ocular inflammation. Methods. Using ELISA systems recently developed, sFasL concentrations in aqueous humor (AH) and/or vitreous fluid (VF) were measured. AH was obtained from 17 eyes of 17 uveitis patients and from 12 eyes of 12 non-uveitis (cataract) patients. VF was obtained from 22 eyes of 22 uveitis patients and 7 eyes of 7 non-uveitis (macular hole) patients. Serum levels of sFasL were also determined. Results. sFasL in AH and VF was below the detection limit of the ELISA systems in all non-uveitis eyes. On the other hand, sFasL was detected in AH from uveitis patients where it measured 367.0 ± 154.7 pg/ml (mean ± SEM). sFasL was also detected in VF from uveitis patients where it measured 1132.2 ± 281.7 pg/ml. None of the sera from patients with or without uveitis contained a detectable level of sFas L. Conclusions. sFasL levels in AH and VF are elevated in the eye during ocular inflammation. Fas-FasL mediated apoptosis may play an important role in the regulation of inflammation during uveitis.

Fluorescent method for albumin assay in human aqueous humour and tear fluid

A new method for albumin fluorophotometry in human ocular fluids (aqueous humour and tear fluid) with a K-35 fluorescent probe is proposed. The method provides measurements of albumin concentration and fluorescence parameters characterising properties of binding centres of the albumin molecule. There was a strong correlation between albumin concentrations determined by fluorescent method proposed by us and by electrophoresis of ocular fluid proteins. This method can be used for the analysis of ocular fluid albumin and the state of the tissue-blood barriers.

A brief history of the lysigenous gland hypothesis

This paper summarizes the early history of the lysigenous gland concept, traces its evolution to recent times, and explores some possible causes of conflicting reports of lysigeny and schizogeny. Secretory cavities and ducts are generally thought to form either through the schizogenous separation of cells or through lysigeny (cell lysis). Gland lysigeny was first proposed in 1857 by Karsten, who believed that plant tissues represent solidifications of humoral fluids and that plant secretions are formed by the resorption of previously solidified cells. The lysigenous gland concept has modernized as our understanding of cytology has improved, but it was established early, from Karsten’s hypothesis, long before the influence of artifacts of specimen preparation was appreciated. Different methods of specimen preparation, including variations in the mounting media used to mount freehand sections, may have caused some of the discrepancies between the findings of lysigenists and schizogenists. Tschirch and Haberlandt promoted the schizolysigeny concept, and believed that the conflicting reports resulted from incomplete observations of a developmental process that included both schizogenous separation of cells and cell lysis to form secretory cavities and ducts. Both lysigeny and schizogeny have been reported in the recent literature, although most reports of lysigeny have been opposed by conflicting observations of schizogeny, and lysigeny may represent a false category of gland development caused by the misinterpretation of artifacts.

Molecular identification and coexpression of galanin and GalR-1 galanin receptor in the human ocular ciliary epithelium: differential modulation of their expression by the activation of alpha2- and beta2-adrenergic receptors in cultured ciliary epithelial cells.

Here we report the coexpression of the neuropeptide galanin and GalR-1 galanin receptors in the human ciliary epithelium, a bilayer of neuroepithelial cells [nonpigmented (NPE) and pigmented (PE)] with neuroendocrine functions, and in a cell line (ODM-2) derived from the NPE cells. Stimulation of ODM-2 cells with phorbol ester [phorbol 12-myristate 13-acetate (PMA)] or forskolin resulted in an up-regulation (two- to threefold) of galanin mRNA expression. Procaterol, a selective beta2-adrenergic agonist, and the catecholamine isoproterenol exerted a long-term down-regulation on galanin mRNA expression when added alone or in combination with PMA or forskolin. These actions exerted by procaterol or isoproterenol were abolished in the presence of ICI 118,551, a selective beta2-adrenergic antagonist. A radioimmunoassay for galanin peptide indicated that galanin or a galanin-like product is present in the human aqueous humor fluid and is accumulated with time in the culture medium of ODM-2 cells. It is interesting that norepinephrine, which exhibited no effect on galanin mRNA expression, induced a down-regulation in the level of galanin or galanin-like product accumulated in the medium of cultured ODM-2 cells to levels even lower than those induced by beta2-adrenergic agonists. This effect is best explained by the concomitant up-regulation (four- to fivefold) of GalR-1 galanin receptor transcripts induced through the activation of alpha2-adrenergic receptors. These findings support the view that pathways elicited by the activation of alpha2- and beta2-adrenergic receptors influence the expression of galanin and GalR-1 galanin receptors in ciliary epithelial cells.