IL-6 receptor independent stimulation of human gp130 by viral IL-6.

The genome of human herpes virus 8, which is associated with Kaposi's sarcoma, encodes proteins with similarities to cytokines and chemokines including a homologue of IL-6. Although the function of these viral proteins is unclear, they might have the potential to modulate the immune system. For viral IL-6 (vIL-6), it has been demonstrated that it stimulates IL-6-dependent cells, indicating that the IL-6R system is used. IL-6 binds to IL-6R, and the IL-6/IL-6R complex associates with gp130 which dimerizes and initiates intracellular signaling. Cells that only express gp130 but no IL-6R cannot be stimulated by IL-6 unless a soluble form of the IL-6R is present. This type of signaling has been shown for hematopoietic progenitor cells, endothelial cells, and smooth muscle cells. In this paper we show that purified recombinant vIL-6 binds to gp130 and stimulates primary human smooth muscle cells. IL-6R fails to bind vIL-6 and is not involved in its signaling. A Fc fusion protein of gp130 turned out to be a potent inhibitor of vIL-6. Our data demonstrate that vIL-6 is the first cytokine which directly binds and activates gp130. This property points to a possible role of this viral cytokine in the pathophysiology of human herpes virus 8.

Enzymatically degraded, nonoxidized LDL induces human vascular smooth muscle cell activation, foam cell transformation, and proliferation.

BACKGROUND: Enzymatic, nonoxidative modification transforms LDL to an atherogenic molecule (E-LDL) that activates complement and macrophages and is present in early atherosclerotic lesions. METHODS AND RESULTS: We report on the atherogenic effects of E-LDL on human vascular smooth muscle cells (SMC). E-LDL accumulated in these cells, and this was accompanied by selective induction of monocyte chemotactic protein-1 in the absence of effects on the expression of interleukin (IL)-8, RANTES, or monocyte inflammatory proteins-1alpha and -beta). Furthermore, E-LDL stimulated the expression of gp130, the signal-transducing chain of the IL-6 receptor (IL-6R) family, and the secretion of IL-6. E-LDL invoked mitogenic effects on SMC through 2 mechanisms. First, an autocrine mitogenic circuit involving platelet-derived growth factor and fibroblast growth factor-beta was induced. Second, upregulation of gp130 rendered SMC sensitive to transsignaling through the IL-6/sIL-6R activation pathway. Because E-LDL promoted release of both IL-6 and sIL-6R from macrophages, application of macrophage cell supernatants to prestimulated SMC provoked a pronounced and sustained proliferation of the cells. CONCLUSIONS: E-LDL can invoke alterations in SMC that are characteristic of the evolving atherosclerotic lesion.

Complement and atherogenesis: binding of CRP to degraded, nonoxidized LDL enhances complement activation.

Complement activation occurs in temporal correlation with the subendothelial deposition of LDL during early atherogenesis, and complement also plays a pathogenetic role in promoting lesion progression. Two lesion components have been identified that may be responsible for complement activation. First, enzymatic degradation of LDL generates a derivative that can spontaneously activate complement, and enzymatically degraded LDL (E-LDL) has been detected in the lesions. Second, C-reactive protein (CRP) colocalizes with complement C5b-9, as evidenced by immunohistological studies of early atherosclerotic lesions, so the possibility exists that this acute phase protein also fulfills a complement-activating function. Here, we report that addition of LDL and CRP to human serum did not result in significant C3 turnover. Addition of E-LDL provoked complement activation, which was markedly enhanced by CRP. Binding of CRP to E-LDL was demonstrated by sucrose flotation experiments. Binding was Ca(2+)-dependent and inhibitable by phosphorylcholine, and the complement-activating property of E-LDL was destroyed by treatment with phospholipase C. These results indicated that CRP binds to phosphorylcholine groups that become exposed in enzymatically degraded LDL particles. Immunohistological studies complemented these findings in showing that CRP colocalizes with E-LDL in early human atherosclerotic lesions. Thus enzymatic, nonoxidative modification of tissue-deposited LDL can be expected to confer CRP-binding capacity onto the molecule. The ensuing enhancement of complement activation may be relevant to the development and progression of the atherosclerotic lesion.

Novel path to activation of vascular smooth muscle cells: up-regulation of gp130 creates an autocrine activation loop by IL-6 and its soluble receptor.

This study describes a novel path to the activation of smooth muscle cells (SMC) by the IL-6/soluble IL-6 receptor (sIL-6R) system. Human vascular SMC constitutively express only scant amounts of IL-6R and so do not respond to stimulation with this cytokine. We show that SMC also do not constitutively express appreciable levels of gp130, which would render them sensitive to transsignaling by the IL-6/sIL-6R complex. Because gp130 is generally believed not to be subject to regulation, SMC would thus appear not to qualify as targets for the IL-6/sIL-6R system. However, we report that treatment of SMC with IL-6/sIL-6R provokes marked up-regulation of gp130 mRNA and surface protein expression. This is accompanied by secretion of IL-6 by the cells, so that an autocrine stimulation loop is created. In the wake of this self-sustaining system, there is a selective induction and secretion of MCP-1, up-regulation of ICAM-1, and marked cell proliferation. The study identifies SMC as the first example of cells in which gp130 expression is subject to substantive up-regulation, and discovers a novel amplification loop involving IL-6 and its soluble receptor that drives SMC into a proinflammatory state.

Enzymatically modified, nonoxidized LDL induces selective adhesion and transmigration of monocytes and T-lymphocytes through human endothelial cell monolayers.

Circulating monocytes and T lymphocytes extravasate through the endothelium at sites of developing atheromatous lesions, where they tend to accumulate and mediate the progression of the disease. We have previously demonstrated the presence of an enzymatically degraded, nonoxidized form of LDL (E-LDL) in early human fatty streaks, which possesses major biological properties of an atherogenic lipoprotein. The effects of E-LDL on human endothelial cells have now been studied with respect to adhesion and transmigration of monocytes and T lymphocytes. E-LDL induced a rapid and dose-dependent selective adhesion of monocytes and T lymphocytes to endothelial cell monolayers within 30 minutes of incubation. Maximal increases in the number of adherent monocytes (8-fold) and of adherent T lymphocytes (4-fold) were observed after treatment with 50 microg/mL E-LDL. E-LDL was more active than oxidized LDL (ox-LDL), whereas native LDL produced only minor adhesive effects. Both E-LDL and ox-LDL enhanced transmigration of monocytes and of T lymphocytes through endothelial monolayers. Again, E-LDL was more potent than ox-LDL, inducing transmigration to a similar extent as N-formyl-Met-Leu-Phe. In endothelial cells, E-LDL stimulated upregulation of intercellular adhesion molecule-1 (ICAM-1), platelet-endothelial cells adhesion molecule-1 (PECAM-1), P-selectin, and E-selectin with distinct kinetics. Analyses with blocking antibodies indicated that ICAM-1 and P-selectin together mediated approximately 70% of cell adhesion, whereas blocking of PECAM-1 had no effect on adhesion but reduced transmigration to less than 50% of controls. E-LDL also upregulated expression of ICAM-1 in human aortic smooth muscle cells, and this correlated with increased adhesion of T lymphocytes. E-LDL is thus able to promote the selective adhesion of monocytes and T lymphocytes to the endothelium, stimulate transmigration of these cells, and foster their retention in the vessel wall by increasing their adherence to smooth muscle cells. These findings underline the potential significance of E-LDL in the pathogenesis of atherosclerosis.

Atherogenic properties of enzymatically degraded LDL: selective induction of MCP-1 and cytotoxic effects on human macrophages.

The mechanisms underlying the selective accumulation of macrophages in early atherosclerotic lesions are poorly understood but are likely to be related to specific properties of altered low density lipoprotein (LDL) deposited in the subendothelium. Enzymatic, nonoxidative degradation of LDL converts the lipoprotein to a potentially atherogenic moiety, enzymatically altered LDL (E-LDL), which activates complement and is rapidly taken up by human macrophages via a scavenger receptor-dependent pathway. Immunohistological evidence indicates that E-LDL is present in an extracellular location in the early lesion. We report that E-LDL causes massive release of monocyte chemotactic protein 1 (MCP-1) from macrophages and that expression of interleukin 8 or RANTES remains unchanged. Release of MCP-1 was preceded by a rapid expression of MCP-1 mRNA, which was detectable after 15 minutes, reached maximum levels after 1 hour, and remained detectable for 12 hours after exposure to concentrations as low as 10 microg/mL E-LDL. MCP-1 mRNA induction and protein release by E-LDL exceeded that evoked by oxidized LDL. Release of MCP-1 was dependent on de novo protein synthesis and on the activity of tyrosine kinases. At higher concentrations, E-LDL, but not oxidized LDL, exerted toxic effects on macrophages that in part appeared to be due to apoptosis. The results show that E-LDL possesses major properties of an atherogenic lipoprotein.

Langerhans cell histiocytosis in children: does soluble interleukin-2-receptor correlate with both disease extent and activity?

BACKGROUND: Langerhans cell histiocytosis (LCH) is characterized by monoclonal proliferation of activated Langerhans cells. Neither etiology nor pathomechanism of this disorder is presently known. However, despite monoclonality LCH might represent a reactive clonal disorder induced by immune dysfunction rather than a malignant process. To investigate a putative cytokine dysregulation in the pathogenesis of this disorder and searching for parameters of both disease activity and prognosis, serum concentrations of proinflammatory and T-cell derived cytokines were evaluated in LCH patients. MATERIALS AND METHODS: Serum levels of IL-1 beta, IL-2, sIL-2R and TNF-alpha were determined by ELISA in seven children with different types of LCH: Three children (aged 6, 10 and 14 years, respectively) with single system/single bone disease; one child (11 years) with recurrent single system/multiple bone disease and three children (1, 2 and 2 years, respectively) with multisystem disease. RESULTS: sIL-2R was elevated at diagnosis in seven children as compared to healthy adults (mean +/- SEM: 5,256 +/- 3,751 U/ml vs. 73 +/- 5.5 U/ml; P < 0.005) or healthy children (mean +/- SEM: 10,195 +/- 2,798 pg/ml vs. 2,638 +/- 156 pg/ml; P < 0.01). A positive correlation between serum levels of sIL-2R and extent of the disease could be observed. During remission, sIL-2R levels declined. IL-1 beta, IL-2, and TNF-alpha remained within the normal range during the study period. CONCLUSIONS: Elevated sIL-2R levels seem to correlate positively with both extent and activity of LCH, thus indicating a pathological T-cell activation as a pathogenetic factor. sIL-2R level is a promising parameter to monitor disease activity in LCH and may also be of prognostic relevance.

Immunohistochemical demonstration of enzymatically modified human LDL and its colocalization with the terminal complement complex in the early atherosclerotic lesion.

Treatment of low density lipoprotein (LDL) with degrading enzymes transforms the molecule to a moiety that is micromorphologically indistinguishable from lipoproteinaceous particles that are present in atherosclerotic plaques, and enzymatically modified LDL (E-LDL), but not oxidized LDL (ox-LDL), spontaneously activates the alternative complement pathway, as do lesion lipoprotein derivatives. Furthermore, because E-LDL is a potent inducer of macrophage foam cell formation, we propose that enzymatic degradation may be the key process that renders LDL atherogenic. In this article, we report the production of two murine monoclonal antibodies recognizing cryptic epitopes in human apolipoprotein B that become exposed after enzymatic attack on LDL. One antibody reacted with LDL after single treatment with trypsin, whereas recognition by the second antibody required combined treatment of LDL with trypsin and cholesterol esterase. In ELISAs, both antibodies reacted with E-LDL produced in vitro and with lesion complement activator derived from human atherosclerotic plaques, but they were unreactive with native LDL or ox-LDL. The antibodies stained E-LDL, but not native LDL or ox-LDL, that had been artificially injected into arterial vessel walls. With the use of these antibodies, we have demonstrated that early human atherosclerotic coronary lesions obtained at autopsy as well as lesions examined in freshly explanted hearts always contain extensive extracellular deposits of E-LDL. Terminal complement complexes, detected with a monoclonal antibody specific for a C5b-9 neoepitope, colocalized with E-LDL within the intima, which is compatible with the proposal that subendothelially deposited LDL is enzymatically transformed to a complement activator at the earliest stages in lesion development.

Endocytosis, storage, and release of IgE by human platelets: differences in patients with type I allergy and nonatopic subjects.

Platelets of atopic individuals differ in alpha-granular contents and in the amount of biologically active mediators released compared with platelets of nonatopic subjects. Because platelets carry the low-affinity IgE receptor (CD23), they may contribute to long-lasting IgE sensitivity by serving as a storage pool for IgE. We compared 45 atopic individuals with immediate-type allergies and 25 nonatopic control subjects with respect to storage and release of IgE by their platelets. Platelets of atopic individuals were characterized by a 10-fold higher median IgE content compared with those of nonatopic control subjects. The platelet IgE content correlated with the serum IgE level in the four atopic individuals with seasonal allergies who were followed up monthly over 1 year. Platelet stimulation with platelet activating factor, but not with thrombin or adenosine diphosphate, resulted in a release of 65% of the stored IgE. Conversely, platelet stimulation with monoclonal IgE/kappa resulted in the release of the chemokine RANTES. Platelet alpha-granules were identified as the main storage compartment for IgE by postembedding immunocytochemistry. Although more than half of the alpha-granules showed gold labeling for IgE, additional labeling was found on the external face of the plasma membrane and within the open canalicular system, indicating endocytosis and exocytosis of IgE. Moreover, the detection of CD23 not only on the plasma membrane but also on membranes of the alpha-granules further supports the existence of an exchange of IgE between the blood plasma and an internal storage compartment. Endocytosis could be confirmed by the uptake of an IgE myeloma protein coupled to colloidal gold. We conclude that platelets of atopic individuals may contribute to allergic inflammation by serving as a storage pool for IgE and by their increased capacity to liberate further mediators of allergy in response to IgE stimulation.

Low efficiency of active immunization against diphtheria in chronic hemodialysis patients.

Recent epidemiological studies indicate a low immunity to diphtheria in adults in industrialized countries. In the light of the epidemic increase of diphtheria in countries such as Russia and the Ukraine, systematic vaccination against this disease is recommended. We analyzed the immunity to diphtheria of 228 hemodialysis patients and the efficiency of single versus triple vaccination against diphtheria. Antibodies against diphtheria toxoid were determined by enzyme immunoassay in sera of 228 adult hemodialysis patients. Fifty-four patients were triple vaccinated against diphtheria and were followed for six months; 17 patients were single immunized and antitoxoid titers were determined 1 and 12 months later. The overall protection rate against diphtheria was 22% and equal in male and female patients. After triple immunization, only 35% of the patients developed protective antibody concentration (> 0.1 i.e./ml) six months after the third vaccination. A single vaccination caused protective titres twelve months later in 41% of the patients. There was no difference between responders and non-responders in the duration, intensity or modality of hemodialysis treatment or the response to previous vaccinations against hepatitis-B. We suggest to monitor antibodies against diphtheria toxoid in vaccinated hemodialysis patients at risk for diphtheria since protective titers are often not attained by the standard vaccination protocol.

Subclass typing of IgG paraproteins by immunofixation electrophoresis.

We present a simple method for subclass typing of IgG paraproteins, with which we have demonstrated a large number of paraproteins that were undetected by conventional immunofixation techniques. The types and distribution of IgG subclass paraproteins were analyzed in 92 human sera in which IgG paraproteins had been demonstrated. The IgG subclass paraproteins were separated by agarose gel electrophoresis rapidly and simply and then typed with the use of sheep anti-human monospecific IgG1-IgG4 antibodies. In 24 of the sera analyzed, IgG subclass typing revealed 25 additional monoclonal bands that were not detected by conventional immunofixation electrophoresis with anti-IgG antisera. Most of these belonged to a different subclass type. The overall subclass frequencies were 68% IgG1, 13% IgG2, 16% IgG3, and 3% IgG4. The distribution of paraprotein subclasses, however, was different in monoclonal gammopathies of undetermined significance in which more IgG3 was shown, whereas in non-Hodgkin lymphomas the number of IgG2 paraproteins was greater than expected; this finding may have diagnostic and prognostic significance.

Low prevalence of diphtheria antitoxin in children and adults in northern Germany.

Recent outbreaks of diphtheria in neighbouring eastern European countries and in the Russian Federation prompted us to evaluate immunity to diphtheria in a sample of 400 healthy individuals (210 male, 190 female) from northern Germany. An age-stratified study population was chosen, including newborns, children, adults and elderly persons over 60 years divided into 8 subgroups of 50 persons each. Diphtheria antitoxin was tested by enzyme immunoassay. The median antitoxin titre was 0.39 IU/ml. There was no difference in the median antitoxin titres of men and women. Inadequate immunity to diphtheria was detected in more than 90% of the 400 individuals tested, including 4% who completely lacked immunity (titre < 0.01 IU/ml), a further 20% with minimal protection (titre 0.01-0.1 IU/ml) and the majority of 69% who showed relative protection for less than one year (titre 0.1-1.0 IU/ml). Only 7% exhibited lasting protection for more than five years (titre > 1.1). Newborns and persons over 50 years of age constituted the least protected groups, with significantly lower median antitoxoid titres than the other age groups (p < 0.001). The absence of protective immunity in 7 of the 50 newborns examined (14%) reflects the inadequate protection of women of reproductive age. Children aged 1 to 10 years were the best immunized and protected group. The results suggest that routine booster immunizations of the majority of the adult population would be advisable in view of the ongoing migration from and the visits to high-risk areas.

Antiproliferative and recovery effects during treatment of breast and ovarian carcinoma cell cultures with interferon-gamma.

We studied the antiproliferative effects of human interferon-gamma (IFN-gamma) on cell lines derived from human carcinomas (three breast, two ovarian, and one renal) and recovery from these effects when IFN-gamma was removed after 6 or 72 h. IFN-gamma led to a dose-dependent and time-dependent cytostatic inhibition of all six tumor cell lines; the renal carcinoma cells were by far the most sensitive, and with these, cytotoxic effects were also seen. The 50% inhibitory dose (ID50) for each cell line was different and remarkably constant over many months. When cells were exposed to IFN-gamma for only 9 or 72 h, those from three lines recovered completely from the growth inhibitory effects, but from three only partially. When cultured for several weeks in the presence of 1600 U/ml of IFN-gamma, two lines developed increased resistance to IFN-gamma, one became much less sensitive, and two showed no changes in sensitivity. We saw no correlations between these changes during continuous exposure to IFN-gamma and the antiproliferative ID50 for each cell or whether the cells recovered completely from the inhibitory effects of IFN-gamma after short-term exposure. Nevertheless, cells with a population doubling time of less than 48 h had low to moderate sensitivity to IFN-gamma and seemed to recover more completely than those doubling in more than 61 h. Our results indicate great individual variation in the in vitro sensitivity of carcinoma cells to the antiproliferative effects of IFN-gamma.

[Intestinal vasculitis and glomerulonephritis in hepatitis C- associated cryoglobulinemia]. / Intestinale Vaskulitis und Glomerulonephritis bei Hepatitis-C-assoziierter Kryoglobulinämie.

In a 53-year-old female patient with recurrent, sometimes bloody diarrhea, the long standing diagnosis of an ANA-negative lupus erythematosus with membranoproliferative glomerulonephritis, leucocytoclastic vasculitis and chronic hepatitis was ruled out and the diagnosis of a hepatitis C associated cryoglobulinaemia was established. The origin of the diarrhea was due to intestinal vasculitis as a result of cold food or beverages.

Impact of allergy screening for blood donors: relationship to nonhemolytic transfusion reactions.

There has been some discussion whether the atopic disposition of a blood donor is associated with a potentially higher incidence of hypersensitivity nonhemolytic transfusion reactions (NHTRs). Serum samples from patients who had suffered from NHTRs and samples from the platelet concentrates (PCs) responsible for the reactions were examined for total and specific IgE as diagnostic markers for allergic events. In addition, the allergy prevalence among 1,088 blood donors was determined to analyze the allergy prevalence among our blood donors. Our results indicate that in 90% of cases, allergic NHTRs were associated with specific IgE antibodies in the recipient's serum, indicating the allergic disposition of the patient. In contrast, specific IgE antibodies were detected in only 22% in the transfused PCs. However, among all investigated NHTRs, there was not a single case in which specific IgE antibodies were detected exclusively in the PC. The allergy prevalence among our blood donors was about 26%. In our opinion, the few cases in which the allergic disposition of blood donors in combination with the allergic disposition of the recipients was associated with NHTRs reflects the allergy prevalence among our blood donors in general (26%). On the basis of these findings, we conclude that allergy diagnosis for blood donors is only of minor value in the prevention and prediction of NHTRs, whereas allergy diagnosis for patients who require multiple PC transfusion might be helpful.

Ifosfamide and carboplatin combined with 41.8 degrees C whole-body hyperthermia in patients with refractory sarcoma and malignant teratoma.

The purpose of this study was to evaluate the pharmacokinetics, biological interactions, and toxicities of ifosfamide and carboplatin combined with 41.8 degrees C whole-body hyperthermia (WBH) for 1 h in a pilot clinical study. Nineteen patients with refractory sarcoma or malignant teratoma were treated. To obtain baseline pharmacokinetic data for ifosfamide, the first chemotherapy course was given without WBH in six patients. This enabled comparison of systemic toxicity and pharmacokinetics of the drug combination with and without WBH (+/- WBH). All other patients received three thermochemotherapy treatments every 3 weeks. Ifosfamide was escalated from 5 to 10 g/m2 with a fixed carboplatin dose of 480 mg/m2. WBH was induced by extracorporally heated blood (in a hemodialysis apparatus) with general anesthesia. The drugs were given at target temperature. A total of 49 thermochemotherapy treatments was administered. The use of the hemodialysis device resulted in an approximate one-third reduction of blood concentrations of 4-hydroxyifosfamide, one activated intermediate metabolite of ifosfamide and carboplatin, but in an increase of chloroacetaldehyde, the other main ifosfamide metabolite. The WBC counts and the platelet nadirs (up to WBH grade 4) were not significantly different +/- WBH. Of 19 evaluable patients, 7 partial remissions, 8 disease stabilizations (average duration, 3 months), and 4 patients with progressive disease were observed. There was no WBH-related mortality. Toxicities observed included mild (anasarca, diarrhea, pressure sores, and perioral herpes simplex) and severe (reversible neuropathy, cardiopulmonary distress, and severe renal dysfunction). No hepatic or central nervous system toxicity occurred. Nephropathy was the dose-limiting toxicity. In conclusion, ifosfamide and carboplatin can be administered with extracorporally induced WBH with acceptable toxicity. Results obtained are consistent with continued evaluation of this combined modality approach.

Expression of sCD23 in atopic and nonatopic blood donors: correlation with age, total serum IgE, and allergic symptoms.

Although structure, biologic activities, and expression of the low-affinity IgE receptor (FceRII, CD23) have been investigated, the diagnostic value for allergies of this molecule and its soluble circulating fragment (sCD23) remains unclear. Therefore, serum sCD23 levels were measured in 203 blood donors. They were divided into atopic and nonatopic subjects by allergy history, physical findings of allergic symptoms, and corresponding specific circulating IgE antibodies. The group consisting of nonatopic subjects was divided into four age categories in order to exclude age-dependent variations in the expression of the low-affinity IgE receptor. In our study population, sCD23 serum levels were not influenced by age. Furthermore, no significant differences, especially no decrease in serum sCD23 levels, between the four nonatopic age groups were detected. There was no significant increase of sCD23 serum levels in atopic subjects in comparison with nonatopic blood donors. In addition, no correlation between total IgE levels and sCD23 serum levels could be detected, in either the group of atopic donors or the group of nonatopics. Our data suggest that the circulating low-affinity IgE receptor does not appear to be an additional general marker for the diagnosis of allergies, as previously suggested.

[Cytokines, immunoglobulins, and IgG subclasses in patients with IgG plasmacytomas]. / Zytokine, Immunglobuline und IgG-Subklassen bei Patienten mit IgG-Plasmozytomen.

Cytokines play an essential role in normal and malignant B-cell proliferation and isotype-switching. Therefore we determined serum levels of different B-cell activating cytokines including IFN gamma, IL-4 and IL-10, IgG subclasses, further immunoglobulins and the soluble B-cell activation marker sCD23 in 68 patients with recently diagnosed and previously untreated IgG myelomas in comparison with age- and sex-matched healthy controls. Our results demonstrated that 16% of the myeloma patients had elevated IL-10 levels up to 1000 pg/ml and 8% showed increased IFN gamma serum concentrations. By contrast, only 7% of the controls showed detectable IL-10 levels and 3% had measurable IFN gamma levels. While in men 67% of the paraproteins were of the kappa light chain type, we found an equal occurrence of kappa and lambda light chains in women. In addition, the distribution of IgG subclasses differed in men and women. In comparison with the controls, no alteration of sCD23 was detected in myeloma patients. We found an apparent correlation of elevated IL-10 levels and the IgG1 subclass.