Ann Chernosky

Selective chemokine mRNA accumulation in the rat spinal cord after contusion injury

Following traumatic injury to the spinal cord, hematogenous inflammatory cells including neutrophils, monocytes, and lymphocytes infiltrate the lesion in a distinct temporal sequence. To examine potential mechanisms for their recruitment, we measured chemokine mRNAs in the contused rat spinal cord, using specific and sensitive reverse transcriptase polymerase chain reaction (RT‐PCR) dot‐blot hybridization assays. The neutrophil chemoattractant GRO‐α was 30‐fold higher than control values at 6 hr postinjury and decayed rapidly thereafter. LIX, a highly related α‐chemokine, also was elevated early postinjury. Monocyte chemoattractant peptide (MCP)‐1 and MCP‐5 mRNAs, potent chemoattractants for monocytes, were significantly elevated at the lesion epicenter at 12 and 24 hr postinjury and declined thereafter. Interferon‐γ‐inducible protein, 10 kDa (IP‐10), chemoattractant towards activated T‐lymphocytes, was significantly elevated at 6 and 12 hr postinjury. The dendritic cell chemoattractant MIP‐3α also was increased, perhaps contributing to the development of T‐cell autoreactivity to neural components after spinal cord injury (SCI) in rats. Other β‐chemokines, including MIP‐1α and RANTES (regulated on expression normal T‐cell expressed and secreted), were minimally affected by SCI. Expression of chemokines, therefore, directly precedes the influx of target neutrophils, monocytes, and T‐cells into the spinal cord postinjury, as noted previously. Thus, selective chemokine expression may be integral to inflammatory processes within the injured spinal cord as a mechanism of recruitment for circulating leukocytes. J. Neurosci. Res. 53:368–376, 1998.

Treatment with BBB022A or rolipram stabilizes the blood-brain barrier in experimental autoimmune encephalomyelitis: an additional mechanism for the therapeutic effect of type IV phosphodiesterase inhibitors

We examined the treatment effects of two structurally distinct phosphodiesterase type IV (PDE IV) inhibitors, BBB022 and rolipram, in murine and rat models of experimental autoimmune encephalomyelitis (EAE). Based on our data, we propose a mechanism of action which may supplement immunomodulatory effects of PDE IV inhibitors. In particular, PDE inhibitors promote elevation of intracellular cAMP levels, increasing the electrical resistance of endothelial monolayers by stabilizing intercellular junctional complexes. Such an effect on central nervous system (CNS) vascular endothelium has the potential to reduce disease severity in EAE, because both inflammatory cells and humoral factors readily cross a disrupted blood-brain barrier (BBB). In this report, we demonstrate the capacity of BBB022 and rolipram to decrease clinical severity of EAE. further, PDE IV inhibitors significantly reduced BBB permeability in the spinal cords of mice with EAE. These results provide evidence that PDE IV-inhibitors may exert therapeutic effects in EAE by modifying cerebrovascular endothelial permeability, reducing tissue edema as well as entry of inflammatory cells and factors.

Apolipoprotein A-I in bile inhibits cholesterol crystallization and modifies transcellular lipid transfer through cultured human gall-bladder epithelial cells

Background: Apolipoprotein A‐I (Apo A‐I), conventionally purified by several steps including organic solvent‐delipidation from plasma, inhibits cholesterol crystallization in bile. To observe a significant effect in vitro, however, supraphysiological concentrations above 100 μg/mL are required. For this reason, this protein has not been considered to play a physiological role in vivo. In the present study, we examined the cholesterol crystal growth‐inhibiting effect of biliary Apo A‐I at its physiological concentration, the modification of transcellular transfer of biliary lipids through cultured human gall‐bladder epithelial cells (GBEC) by Apo A‐I at its physiological concentration and the binding and secretion of Apo A‐I by GBEC.

CHEMOKINES AND CHEMOKINE RECEPTORS IN MODEL NEUROLOGICAL PATHOLOGIES : MOLECULAR AND IMMUNOCYTOCHEMICAL APPROACHES

Publisher Summary Chemokines are proposed to play a role in CNS inflammatory disease at this stage of leukocyte recruitment. In this sense, chemokines exert an essential function in the development of immune-mediated CNS inflammation. This concept is supported by several studies of CNS chemokine expression during experimental autoimmune encephalomyelitis (EAE), all of which indicate an intimate relationship between chemokine expression and clinical disease. Further, antibodies to one chemokine macrophage inflammatory protein-1α, reduces the severity of passive-transfer EAE. Inflammatory cells enter nervous system tissues in virtually all pathological states, with the character of the infiltrate differing widely, according to the pattern of tissue injury. This chapter studies chemokine production in the CNS during several models of nervous system pathology. These studies, in aggregate, begin to sketch an intriguing relationship between the preferential expression of individual chemokines and the recruitment of specific leukocyte cell types to the CNS or peripheral nervous system (PNS) compartment.

Human Gallbladder Mucosal Function (Effects on Intraluminal Fluid and Lipid Composition in Health and Disease)

Gallbladder mucosal absorption of fluid duringfasting is a well-known process. Indirect in vivo andrecent in vitro evidence for physiologically relevantgallbladder absorption of cholesterol and phospholipids from bile has been observed in humans. Thepresent study explored and compared by indirect meansthe relative efficiences of human gallbladder mucosalabsorption of fluid and lipids in health and disease. Biliary lipids and pigment content weremeasured in fasting gallbladder bile samples obtainedfrom gallstone-free controls and from four study groups:multiple and solitary cholesterol gallstone patients, and morbidly obese subjects with and withoutgallstones. Bile salts and pigment content weresignificantly greater in gallstone-free controls than inall other disease study groups. This was interpreted as evidence of more effective gallbladdermucosal fluid absorption in nonobese gallstone-freecontrols compared to that in all other groups.Correlation plot analyses of biliary lipids showed lowerconcentrations of phospholipids than expected from the indexbile salt concentrations. The same was found forcholesterol concentrations but only in supersaturatedsamples. These findings were much more pronounced in gallstone free-controls and were accordinglyinterpreted as evidence of more efficient gallbladderabsorption of both phospholipids and cholesterol incontrols compared with that found in each of the disease study groups. Moreover, impaired gallbladdermucosal function, while invariably associated withcholesterol gallstone disease, was not found to be anecessary consequence of the physical presence ofstones. It is concluded that efficient gallbladdermucosal absorption of both fluid and apolar lipids frombile is a normal physiological process that is oftenseriously impaired in the presence of either cholesterol gallstone disease or at least one of itsprecursor forms.

Purification and characterization of a novel human 15 kd cholesterol crystallization inhibitor protein in bile

Crystallization-inhibiting proteins can explain longer nucleation times associated with bile from gallstone-free subjects as compared with bile from patients with cholesterol gallstones. We partially characterized and examined the crystallization inhibitory potency of a newly purified 15 kd human biliary protein. Gallbladder bile was passed through an anti-apolipoprotein A-I (apo A-I) immunoaffinity column to extract lipid-associated proteins. The bound fraction was separated by 30 kd ultrafiltration. Sodium dodecyl sulfate-polyacrylamide gel electrophesis (SDS-PAGE) was performed under nonreducing and reducing conditions. Cholesterol crystallization activity was tested in a photometric cholesterol crystal growth assay. Isoelectric focusing was performed by using a standard gel. The purified 15 kd protein was subjected to N-terminal amino acid sequencing. Although the whole apo A-I-bound fraction contained a variety of proteins and lipids, its 30 kd filtrate yielded a nearly pure 15 kd protein with only minor contamination from apo A-1. Amino acid sequencing showed that the protein was unique. Enzymatic deglycosylation revealed no evidence for glycosylation. At a protein concentration of 10 micrograms/ml, crystallization time was delayed as compared with control and apo A-I, and final crystal mass was reduced to 75% of control. Its isoelectric point was 6.1 without isoforms. Under nonreducing conditions, the protein formed a 30 kd dimer and a 60 kd tetramer. We conclude that this protein is a novel potent biliary crystallization inhibitor protein.

Comparison of haptoglobin and apolipoprotein A‐I on biliary lipid particles involved in cholesterol crystallization

Several proteins are known to modulate cholesterol crystallization. We recently demonstrated that haptoglobin has cholesterol crystallization promoting activity. However, this effect is still not well understood mechanistically. The current study examined the distribution of haptoglobin compared to apolipoprotein A‐I (apo A‐I) to micelles, vesicles and crystals as an initial step in providing a focus for further studies of the mechanism of cholesterol crystallization activity. Specific protein purification was accomplished by immunoaffinity chromatography. The crystallization‐promoting activity of biliary haptoglobin, albumin and commercial apo A‐I was measured by a photometric crystal growth assay. The distribution of micelles, vesicles and proteins in model bile was determined by Sepharose CL‐6B column chromatography. Detection of the presence of test proteins in cholesterol crystals was determined using specific 125I‐radiolabelled proteins. Haptoglobin (20 μg/mL) showed a significant crystallization promoting‐activity, whereas apo A‐I (30 μg/mL) only tended to show a slight inhibitory activity. The cholesterol crystal‐bound protein in each case was found to be less than 1% of the total concentration of that protein that had been added to the model bile system. The elution profile of commercial apo A‐I from a Sepharose CL‐6B column was strikingly altered when it was added to model bile prior to elution. In contrast, the column elution profiles for both haptoglobin and albumin were unchanged when model bile was similarly added to the sample. Haptoglobin increased the amount of cholesterol found in the vesicular fraction when compared to apo A‐I. Haptoglobin does not bind tightly to either biliary lipid particles or to cholesterol crystals but does increase the amount of cholesterol in vesicles by inducing a shift from micellar cholesterol (P=0.046). This shift appears to explain in part its promoting effect on cholesterol crystallization.

Variations in Pigment and Carbohydrate Content of Gallbladder Bile Affect Accurate Quantitation of Total Protein When Using the Fluorescamine Method

BACKGROUND Despite solute dilution and reduced total lipid concentrations, an unexplained increase in protein concentration has been reported to occur in the gallbladder bile of cholesterol gallstone patients. METHODS Solutes in gallbladder bile from gallstone-free controls and from four study groups were measured using standard methods. Total proteins were measured using amino acid analysis and a conventional fluorescamine method. RESULTS Bile salts and pigment content were greater in gallstone-free controls than in all other study groups, including morbidly obese gallstone-free subjects. Total biliary protein concentration, as determined by amino acid analysis in the gallstone-free control group was higher than in non-obese gallstone patients with multiple stones and in morbidly obese gallstone-free subjects. Total biliary proteins as measured with fluorescamine, however, did not show intergroup differences. A major problem of the conventional fluorescamine assay is shown to be an artefact arising from the high pigment content of the more concentrated samples. CONCLUSIONS Very dilute gallbladder bile samples are often found in the presence of gallstone disease. This also occurs in morbidly obese subjects, even in the absence of gallstones. Although the contribution of protein secretion/absorption by the gallbladder can also be relevant, especially in the presence of morbid obesity, the protein concentration in gallbladder bile, when accurately measured, generally parallels the concentrations of non-absorbed biliary solutes, reflecting the efficiency of fluid absorption. Measurement of biliary proteins by the conventional fluorescamine method is unreliable in clinical studies in which intergroup differences in pigment content are commonly present.