Interleukin (IL) 31 induces in cynomolgus monkeys a rapid and intense itch response that can be inhibited by an IL-31 neutralizing antibody.

BACKGROUND: Overexpression or administration of interleukin 31 (IL-31) has been shown to induce a profound itch response in mice and dogs. The chronic pruritus observed in mouse IL-31 transgenic mice results in the development of skin lesions and alopecia through excoriation from excessive scratching, a condition similar to that observed in patients with atopic dermatitis (AD). OBJECTIVE: To test whether IL-31 induces pruritus in non-human primates and, if so, whether treatment with an anti-IL-31 neutralizing monoclonal antibody (mAb) can block the response. METHODS: A series of studies was conducted in cynomolgus monkeys to evaluate the itch response to recombinant cynomolgus IL-31 (cIL-31) administration. Three routes of cIL-31 administration (intravenous, intradermal, and subcutaneous) were evaluated. Subcutaneous treatment with a humanized anti-human IL-31 mAb cross-reactive to cIL-31 was subsequently tested for its ability to block the response to intradermal cIL-31 administration. RESULTS: Each route of cIL-31 delivery elicited a scratching response immediately after cIL-31 administration and lasted at least 3 h. Treatment with the IL-31 mAb inhibited the cIL-31-mediated scratching response in a dose-dependent manner. CONCLUSION: These results demonstrate that an IL-31 mAb can inhibit IL-31-mediated pruritus in vivo, and could be an effective therapy for pruritic skin conditions like AD where IL-31 upregulation may play a role.

Characterization of apolipoprotein A-I- and A-II-containing lipoproteins in a new case of high density lipoprotein deficiency resembling Tangier disease and their effects on intracellular cholesterol efflux.

A 48-yr-old Caucasian female of central European origin (subject IM) with low plasma cholesterol and normal plasma triglyceride (TG) had extremely low apo A-I (6 mg/dl), A-II (5 mg/dl), and HDL cholesterol (2 mg/dl) levels. She had most of the clinical symptoms typically associated with Tangier disease, including early corneal opacities, yellow-streaked tonsils, hepatomegaly, and variable degrees of peripheral neuropathy, but had no splenomegaly. She had a myocardial infarction at age 46. Since HDL are postulated to be involved in the transport of excess cholesterol from peripheral tissues to the liver for degradation, and the ability of an HDL particle to promote cellular cholesterol efflux appears to be related to its density, size, and apo A-I and A-II contents, we isolated and characterized the HDL particles of this patient and all her first degree relatives (mother, a brother, and two children). The plasma A-I, A-II, and HDL cholesterol levels of all five relatives were either normal or high. Using anti-A-I and anti-A-II immunosorbents, we found three populations of particles in IM: one contained both apo A-I and A-II, Lp(AI w AII); one contained apo A-I but no A-II, Lp(AI w/o AII); and the third (an unusual one) contained apo A-II but no A-I, Lp(AII). Two-thirds of her plasma A-I and A-II existed in separate HDL particles, i.e., in Lp(AI w/o AII) and Lp(AII), respectively. Only Lp(AI w AII) and Lp(AI w/o AII) were present in the plasma of the relatives. All three populations of the patient's HDL particles had a normal core/surface lipid ratio, but the cores were enriched with TG. The apo A-I-containing particles, however, were considerably smaller and contained much less lipid than Lp(AII). Despite these unusual physicochemical characteristics, the apo A-I-containing particles and Lp(AII) were effective suppressors of intracellular cholesterol esterification in cholesterol-loaded human skin fibroblast. The patient's plasma apo D and lecithin cholesterol acyltransferase levels were reduced, with an increased proportion located in non-HDL plasma fractions. These findings are discussed in light of Tangier disease and other known HDL-deficiency cases, and the role of HDL in the maintenance of cell cholesterol homeostasis.

Interaction between high-density lipoprotein subpopulations in apo B-free and abetalipoproteinemic plasma.

Two populations of high-density lipoprotein (HDL) particles exist in human plasma. Both contain apolipoprotein (apo) A-I, but only one contains apo A-II: Lp(AI w AII) and Lp(AI w/o AII). To study the extent of interaction between these particles, apo B-free plasma prepared by the selective removal of apo B-containing lipoproteins (LpB) from the plasma of three normolipidemic (NL) subjects and whole plasma from two patients with abetalipoproteinemia (ABL) were incubated at 37 degrees C for 24 h. Apo B-free plasma samples were used to avoid lipid-exchange between HDL and LpB. Lp(AI w AII) and Lp(AI w/o AII) were isolated from each apo B-free plasma sample before and after incubation and their protein and lipid contents quantified. Before incubation, ABL plasma had reduced levels of Lp(AI w AII) and Lp(AI w/o AII), (40% and 70% of normals, respectively). Compared to the HDL of apo B-free NL plasma, ABL HDL had higher relative contents of free cholesterol, phospholipid and total lipid, and contained more particles with apparent hydrated Stokes diameter in the 9.2-17.0 nm region. These differences were particularly pronounced in particles without apo A-II. Despite their differences, the total cholesterol contents of Lp(AI w AII) increased, while that of Lp(AI w/o AII) decreased in all five plasma samples and the amount of apo A-I in Lp(AI w AII) increased by 6-8 mg/dl in four during the incubation. These compositional changes were accompanied by a relative reduction of particles in the 7.0-8.2 nm Stokes diameter size region and an increase of particles in the 9.2-11.2 nm region. These data are consistent with intravascular modulation between HDL particles with and without apo A-II. The observed increase in apo A-II-associated cholesterol and apo A-I, could involve either the transfer of cholesterol and apo A-I from particles without apo A-II to those with A-II, or the transfer of apo A-II from Lp(AI w AII) to Lp(AI w/o AII). The exact mechanism and direction of the transfer remain to be determined.

Protein transfer between A-I-containing lipoprotein subpopulations: evidence of non-transferable A-I in particles with A-II.

Transfer of apolipoproteins (apo) between the two subpopulations of apo A-I-containing lipoproteins in human plasma: those with A-II [Lp(AI w AII)] and those without [Lp(AI w/o AII)], were studied by observing the transfer of 125I-apo from a radiolabeled subpopulation to an unlabeled subpopulation in vitro. When Lp(AI w AII) was directly radioiodinated, 50.3 +/- 7.4 and 19.5 +/- 7.7% (n = 6) of the total radioactivity was associated with A-I and A-II, respectively. In radioiodinated Lp(AI w/o AII), 71.5 +/- 6.8% (n = 6) of the total radioactivity was A-I-associated. Time-course studies showed that, while some radiolabeled proteins transferred from one population of HDL particles to another within minutes, at least several hours were necessary for transfer to approach equilibrium. Incubation of the subpopulations at equal A-I mass resulted in the transfer of 51.8 +/- 5.0% (n = 4) of total radioactivity from [125I]Lp(AI w/o AII) to Lp(AI w AII) at 37 degrees C in 24 h. The specific activity (S.A.) of A-I in the two subpopulations after incubation was nearly identical. Under similar incubation conditions, only 13.4 +/- 4.6% (n = 4) of total radioactivity was transferred from [125I]Lp(AI w AII) to Lp(AI w/o AII). The S.A. of A-I after incubation was 2-fold higher in particles with A-II than in particles without A-II. These phenomena were also observed with iodinated high-density lipoproteins (HDL) isolated by ultracentrifugation and subsequently subfractionated by immunoaffinity chromatography. However, when Lp(AI w AII) radiolabeled by in vitro exchange with free [125I]A-I was incubated with unlabeled Lp(AI w/o AII), the S.A. of A-I in particles with and without A-II differed by only 18% after incubation. These data are consistent with the following: (1) in both populations of HDL particles, some radiolabeled proteins transferred rapidly (minutes or less), while others transferred slowly (hours); (2) when Lp(AI w AII) and Lp(AI w/o AII) were directly iodinated, all labeled A-I in particles without A-II were transferable, but some labeled AI in particles with A-II were not; (3) when Lp(AI w AII) were labeled by in vitro exchange with [125I]A-I, considerably more labeled A-I were transferable. These observations suggest the presence of non-transferable A-I in Lp(AI w AII).

Effects of pravastatin on apolipoprotein-specific high density lipoprotein subpopulations and low density lipoprotein subclass phenotypes in patients with primary hypercholesterolemia.

UNLABELLED: The HMG-CoA reductase inhibitor class of cholesterol-lowering agents reduces very low density lipoproteins (VLDL) and low density lipoproteins (LDL) and slightly increases high density lipoproteins (HDL). However, the effects of these agents on subclasses within the LDL and HDL fractions are not well understood. We have employed an HMG-CoA reductase inhibitor, pravastatin, to determine if LDL subclass phenotypes, as determined by gradient gel electrophoresis, and HDL particles containing both apolipoprotein (apo) A-I and A-II, Lp(AI w AII), and those containing apo A-I but not A-II, Lp(AI w/o AII) are affected by pravastatin (10 mg daily). Twenty-four subjects with LDL-cholesterol (LDL-C) > 160 mg/dl, triglyceride (TG) < 350 mg/dl and no recent myocardial infarction or secondary causes of hypercholesterolemia were enrolled. Compared with an age- and sex-matched normolipidemic reference group (controls), the hypercholesterolemic subjects had reduced levels of Lp(AI w/o AII) and increased levels of Lp(AI w AII) at baseline. In addition, both of their HDL subpopulations had significantly more small (7.0-8.2 nm) particles (P < 0.02 and 0.0001) but significantly fewer large (9.2-11.2 nm) particles (P < 0.002 and 0.0001). Pravastatin induced statistically significant (P < 0.001) reductions in plasma total C (15%), LDL-C (18%), and apo B (16%). While apo A-I and A-II levels increased 5% (P < 0.001) and 6% (P < 0.05), respectively, concentration, composition, and size abnormalities in Lp(AI w AII) and Lp(AI w/o AII) persisted. Lp(a), apo E and cholesteryl ester transfer protein (CETP) levels also did not change. Although changes in LDL subclass phenotypes were observed, all changes involved the intermediate phenotype, and no significant changes in LDL peak particle diameter were seen in either group. Interrelationships between CETP, LDL subclass phenotypes and HDL subpopulations were also seen. CONCLUSIONS: Although pravastatin decreased plasma apo B and LDL lipid concentrations, no major changes were seen in LDL subclass phenotypes or HDL subpopulations even in the presence of abnormalities associated with arteriosclerosis. Similarly, CETP, which is believed to play a role in HDL and LDL particle size distribution, did not change with pravastatin treatment. Further research is needed to determine the pathophysiological basis of abnormal HDL and LDL subclasses in hypercholesterolemia and explore methods of rectifying the abnormalities.

In vitro transformation of apoA-I-containing lipoprotein subpopulations: role of lecithin:cholesterol acyltransferase and apoB-containing lipoproteins.

Two populations of apoA-I-containing lipoproteins are found in plasma: particles with apoA-II [Lp(AI w AII)] and particles without apoA-II [Lp(AI w/o AII)]. Both are heterogeneous in size. However, their size subpopulation distributions differ considerably between healthy subjects and patients with coronary artery diseases. The metabolic basis for such alterations was studied by determining the role of lecithin:cholesterol acyltransferase (LCAT) and apoB-containing lipoproteins (LpB) in the size subpopulation distributions of Lp(AI w AII) and Lp(AI w/o AII). ApoB-free and LCAT-free plasmas, prepared by affinity chromatography, and whole plasma were incubated at 4 degrees C and 37 degrees C for 24 hr. After incubation, Lp(AI w AII) and Lp(AI w/o AII) were isolated by anti-A-II and anti-A-I immunosorbents. Their size subpopulation distributions were studied by nondenaturing gradient polyacrylamide gel electrophoresis. At 4 degrees C most Lp(AI w AII) particles were in the range of 7.0-9.2 nm Stokes diameter. Incubation of plasma at 37 degrees C resulted in an overall enlargement of particles up to 11.2 nm and larger. These particles were enriched with cholesteryl ester and triglyceride and depleted of phospholipids and free cholesterol. Removal of LpB or LCAT from plasma prior to incubation greatly reduced their enlargement. At 4 degrees C, Lp(AI w/o AII) contained mostly particles of 8.5 and 10.1 nm. Incubation at 37 degrees C abolished both subpopulations with the formation of a new subpopulation of 9.2 nm. This transformation was identical in apoB-free plasma but was not seen in LCAT-free plasma. Our study shows that transformation of Lp(AI w AII) requires both LCAT and LpB. However, LpB is not necessary for the transformation of Lp(AI w/o AII) in vitro. The relevance of these in vitro studies to in vivo lipoprotein metabolism was demonstrated in a subject with hepatic triglyceride lipase deficiency.

Differential effect of ultracentrifugation on apolipoprotein A-I-containing lipoprotein subpopulations.

Two populations of apolipoprotein (apo) A-I-containing lipoprotein particles are found in high density lipoproteins (HDL): those that also contain apo A-II[Lp(A-I w A-II)] and those that do not [Lp(A-I w/o A-II)]. Lp(A-I w/o A-II) comprised two distinct particle sizes with mean hydrates Stokes diameter of 10.5 nm for Lp(A-I w/o A-II)1 and 8.5 nm for Lp(A-I w/o A-II)2. To study the effect of ultracentrifugation on these particles, Lp(A-I w/o A-II) and Lp(A-I w A-II) were isolated from the plasma and the ultracentrifugal HDL (d 1.063-1.21 g/ml fractions) of five normolipidemic and three hyperlipidemic subjects. The size subpopulations of these particles were studied by gradient polyacrylamide gel electrophoresis. Several consistent differences were detected between plasma Lp(A-I w/o A-II) and HDL Lp(A-I w/o A-II). First, in all subjects, the relative proportion of Lp(A-I w/o A-II)1 to Lp(A-I w/o A-II)2 isolated from HDL was reduced. Second, particles larger than Lp(A-I w/o A-II)1 and smaller than Lp(A-I w/o A-II)2 were considerably reduced in HDL. Third, a distinct population of particles with approximate Stokes diameter of 7.1 nm usually absent in plasma was detected in HDL Lp(A-I w/o A-II). Little difference in subpopulation distribution was detected between Lp(A-I w A-II) isolated from the plasma and HDL of the same subject. When plasma Lp(A-I w/o A-II) and Lp(A-I w A-II) were centrifuged, 14% and 4% of A-I were, respectively, recovered in the D greater than 1.21 g/ml fraction. Only 2% A-II was found in this density fraction. These studies show that the Lp(A-I w/o A-II) particles are less stable than Lp(A-I w A-II) particles upon ultracentrifugation. Among the various Lp(A-I w/o A-II) subpopulations, particles larger than Lp(A-I w/o A-II)1 and smaller than Lp(A-I w/o A-II)2 are most labile.

Altered particle size distribution of apolipoprotein A-I-containing lipoproteins in subjects with coronary artery disease.

Plasma high density lipoproteins (HDL) can be separated into two subpopulations of apolipoprotein A-I-containing particles: those that also contain apoA-II [Lp(AI w AII)] and those that do not [Lp(AI w/o AII)]. These particles were isolated by immunoaffinity chromatography from 17 men (9 normolipidemic (NL), 8 hyperlipidemic (HL) with symptomatic coronary artery disease (CAD), from 17 NL men without any symptoms of CAD (healthy controls), and from 10 NL men with entirely normal coronary arteriograms (CAD-free controls). The distributions of particle size in these two subpopulations were determined by gradient gel electrophoresis and densitometric scanning. Approximately half of the Lp(AI w AII) particles in all subjects were distributed in the 8.2-9.2 nm interval. For patients with CAD, a greater fraction of the particles were small, in the 7.0-8.2 nm interval [33% in CAD vs. 26% in CAD-free controls (P less than 0.01) and 19% in healthy controls (P less than 0.0001)], and a smaller fraction of the particles were in the 9.2-11.2 nm interval (14% in CAD vs. 24% in CAD-free control (P less than 0.002) and healthy control groups (P less than 0.001). The Lp(AI w/o AII) of both control groups were primarily composed of two discrete subpopulations in the 8.2-9.2 nm and the 9.2-11.2 nm intervals. In CAD patients there were fewer particles in the 9.2-11.2 nm size interval (23% in CAD vs. 33% in CAD-free controls (P less than 0.005) and 36% in healthy controls (P less than 0.0001), and more particles in the smallest 7.0-8.2 nm size interval (32% in CAD vs. 23% in CAD-free controls (P less than 0.01) and 18% in healthy controls (P less than 0.001]. Thus, the spectrum of HDL particle sizes in patients with CAD tends to be shifted toward the smaller particle when compared with the two control groups. This was observed in both NL and HL patients with HDL cholesterol (CH) values in the normal range. As a group, CAD patients had lower HDL (42 +/- 7 mg/dl) and HDL2 (6 +/- 4 mg/dl) CH than healthy (HDL: 49 +/- 7, HDL2: 12 +/- 6 mg/dl) and CAD-free (HDL: 51 +/- 9, HDL2: 12 +/- 6 mg/dl) controls. When controls and patients were compared for their frequencies of abnormal HDL CH levels and particle sizes, abnormalities in HDL and HDL2 CH levels were not significantly more frequent (twofold) among CAD patients than among controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Does an increase in membrane unsaturated fatty acids account for Tween 80 stimulation of glucosyltransferase secretion by Streptococcus salivarius?

When Streptococcus salivarius was grown in batch culture in the presence of various Tween detergents, the fatty acid moiety of the detergent was incorporated into the lipids of its membrane. Tween 80 (containing primarily oleic acid) markedly stimulated the production of extracellular glucosyltransferase and also increased the degree of unsaturation of the membrane lipid fatty acids. The possibility that an increase in membrane unsaturated fatty acids promoted extracellular glucosyltransferase production was examined by growing cells at different temperatures in the presence or absence of Tween 80. The membrane lipids of cells grown at 30 degrees C, 37 degrees C and 40 degrees C without Tween 80 exhibited unsaturated/saturated fatty acid ratios of 2.06, 1.01 and 0.87 respectively. A significant increase in the production of extracellular glucosyltransferase was observed at 30 degrees C compared to cells grown at 40 degrees C. However, cells produced much more exoenzyme at all temperatures when grown with Tween 80. The results indicated that an increase in the unsaturated fatty acid content of the membrane lipids was not by itself sufficient to account for the stimulation of extracellular glucosyltransferase production by Tween 80, but that the surfactant also had to be present.

Distribution and localization of lecithin:cholesterol acyltransferase and cholesteryl ester transfer activity in A-I-containing lipoproteins.

Two types of A-I-containing lipoproteins are found in human high density lipoproteins (HDL): particles with A-II (Lp(A-I with A-II] and particles without A-II (Lp(A-I without A-II]. We have studied the distribution of lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer (CET) activities in these particles. Lp(A-I with A-II) and Lp(A-I without A-II) particles were isolated from ten normolipidemic subjects by anti-A-I and anti-A-II immunosorbents. Most plasma LCAT mass (70 +/- 15%), LCAT (69 +/- 16%), and CET (81 +/- 15%) activities were detected in Lp(A-I without A-II). Some LCAT (mass: 16 +/- 7%, activity: 17 +/- 8%) and CET activities (7 +/- 8%) were detected in Lp(A-I with A-II). To determine the size subspecies that contain LCAT and CET activities, isolated Lp(A-I with A-II) and Lp(A-I without A-II) particles of six subjects were further fractionated by gel filtration column chromatography. In Lp(A-I without A-II), most LCAT and CET activities were associated with different size particles, with the majority of the LCAT and CET activities located in particles with hydrated Stokes diameters of 11.6 +/- 0.4 nm and 10.0 +/- 0.6 nm, respectively. In Lp(A-I with A-II), most of the LCAT and CET activities were located in particles similar in size: 11.1 +/- 0.4 nm and 10.6 +/- 0.3 nm, respectively. Ultracentrifugation of A-I-containing lipoproteins resulted in dissociation of both LCAT and CET activities from the particles. Furthermore, essentially all CET and LCAT activities were recovered in the non-B-containing plasma obtained by anti-LDL immunoaffinity chromatography. This report, therefore, provides direct evidence for the association of LCAT and CET protein with A-I-containing lipoproteins. Our conclusions pertain to fasting normolipidemic subjects and may not be applicable to hyperlipidemic or nonfasting subjects.

A mutation in the human apolipoprotein A-I gene. Dominant effect on the level and characteristics of plasma high density lipoproteins.

Epidemiologic and genetic data suggest an inverse relationship between plasma high density lipoprotein (HDL) cholesterol and the incidence of premature coronary artery disease. Some of the defects leading to low levels of HDL may be a consequence of mutations in the genes coding for HDL apolipoproteins A-I and A-II or for enzymes that modify these particles. A proband with plasma apoA-I and HDL cholesterol that are below 15% of normal levels and with marked bilateral arcus senilis was shown to be heterozygous for a 45-base pair deletion in exon four of the apoA-I gene. This most likely represents a de novo mutation since neither parent carries the mutant allele. The protein product of this allele is predicted to be missing 15 (Glu146-Arg160) of the 22 amino acids comprising the third amphipathic helical domain. The HDL of the proband and his family were studied. Using anti-A-I and anti-A-II immunosorbents we found three populations of HDL particles in the proband. One contained both apoA-I and A-II, Lp(A-I w A-II); one contained apoA-I but no A-II, Lp(A-I w/o A-II); and the third (an unusual one) contained apoA-II but no A-I. Only Lp(A-I w A-II) and (A-I w/o A-II) were present in the plasma of the proband's parents and brother. Analysis of the HDL particles of the proband by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two protein bands with a molecular mass differing by 6% in the vicinity of 28 kDa whereas the HDL particles of the family members exhibited only a single apoA-I band. The largely dominant effect of this mutant allele (designated apoA-ISeattle) on HDL levels suggests that HDL particles containing any number of mutant apoA-I polypeptides are catabolized rapidly.