Improved methodology for identifying the teratogenic potential in early drug development of hERG channel blocking drugs.

Drugs blocking the potassium current IKr of the heart (via hERG channel-inhibition) have the potential to cause hypoxia-related teratogenic effects. However, this activity may be missed in conventional teratology studies because repeat dosing may cause resorptions. The aim of the present study was to investigate an alternative protocol to reveal the teratogenic potential of IKr-blocking drugs. The IKr blocker astemizole, given as a single dose (80 mg/kg) on gestation day (GD) 13 to pregnant rats caused digital defects. In whole rat embryo culture (2h) on GD 13, astemizole caused a decrease in embryonic heart rate at 20 nM, and arrhythmias at 200-400 nM. Cetirizine, without IKr-blocking properties, did not affect the rat embryonic heart in vitro. The present study shows that single dose testing on sensitive days of development, together with whole embryo culture, can be a useful methodology to better characterize the teratogenic potential of IKr-blocking drugs.

[The Violence Risk Appraisal Guide (VRAG)--a tool for the risk assessment of violent offenders]. / Der Violence Risk Appraisal Guide (VRAG)--ein Instrument zur Kriminalprognose bei Gewaltstraftätern.

OBJECTIVE: Most instruments used for assessing the recidivism risk of an offender with a violent or sex offense have been developed and validated in North America. METHODS: The aim of this study is to discuss the state of validation for the Violence Risk Appraisal Guide (VRAG)--an instrument for assessing the recidivism risk of violent offenders. A systematic literature research forms the basis for the processing of the literature. In a second section, a scientific translation of the instrument to German, including the scoring rules, is presented. RESULTS: Normally, while examining the validity of the VRAG, there is a focus on the discriminatory power (displayed using the so-called Area Under the Curve [AUC]). These examinations showed a satisfactory to good discriminatory power (AUC: 0.70-- 0.86). A standardization of this instrument for populations in Europe respectively the German-speaking area has not yet taken place. Only few studies have verified whether North American standard values are also valid for Europe. The few studies on this subject question the generalizability of these standard values to other countries. CONCLUSIONS: The VRAG can be considered a valid measure for the assessment of recidivism risk in Germany and in Switzerland, although so far, standardization has been dispensed with. The application of the VRAG can provide indications for the evaluation of recidivism risk and be integrated into an individual case-oriented assessment.

In-vitro engineering of implantable human urinary tract tissue matrices.

Due to lack or destruction of functional tissues, congenital and acquired diseases of the genitourinary tract may need, for the engineering of these tissues, biomaterials as cell carriers. Cell cultures of human urothelial and bladder smooth muscle cells were established and growth of the latter, also under mechanical stimulation was analysed. Collagen and polyesterurethane foam scaffolds were evaluated for their aptitude as cell carriers. Scaffolds made of collagens, naturally occurring extracellular matrix proteins, are biocompatible and can induce tissue regeneration and are therefore apt for tissue engineering. The attachment of serum proteins to their surfaces does further improve these characteristics, mimicking an almost natural cell environment. We investigated the aptitude of equine collagen scaffolds (Tissue Fleece) modified by coating foetal bovine serum proteins, for human bladder smooth muscle cell attachment and growth. Furthermore we evaluated a highly porous biodegradable polyesterurethane foam (DegraPol), as scaffold for smooth muscle tissue engineering. These cell-polymer constructs were characterised by histology, scanning electron microscopy, immunohistochemistry and proliferation assays. Cell attachment and growth on collagen scaffolds improved when pre-coated with serum proteins. Cell penetration assessed by histology showed similar results on modified and native scaffolds. Further these cell-scaffold constructs were implanted in the dorsal subcutaneous space of athymic mice. In vivo studies showed the presence of the transplanted smooth muscle cells until day 3. Thereafter angiogenesis was induced and infiltration of mouse fibroblasts and polymorphonuclear cells was observed. Smooth muscle cells grown on DegraPol showed the same morphology as when grown on a control polystyrene surface. Positive immunostaining with anti-alpha smooth muscle actin indicated the preservation of the specific cell phenotype. Micrographs from scanning electron microscopy showed that the cells grew on the foam surface as well as inside the pores. The smooth muscle cells proliferated well on DegraPol, however, proliferation rate decreased due to apoptosis with time in culture. Although the above scaffolds provide an adequate milieu for cell attachment, their ability for cell penetration and growth is reduced. For this reason we are now evaluating a biodegradable poly (ethylene glycol) (PEG) hydrogel, which allows integration of cells within the matrix and also provides an excellent scaffold for the controlled incorporation of biological signals.

Separation of seventeen 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and dibenzofurans and 12 dioxin-like polychlorinated biphenyls by comprehensive two-dimensional gas chromatography with electron-capture detection.

Comprehensive two-dimensional gas chromatography (GC x GC) with electron-capture detection (ECD) has been optimized for the separation of seventeen 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and dibenzofurans and 12 dioxin-like polychlorinated biphenyls, with emphasis on the selection of the first- and second-dimension, commercially available, columns. When eight second-dimension columns were subsequently combined with a 100% methylpolysiloxane stationary phase (DB-1) in the first dimension to create orthogonal conditions, a complete separation of all congeners with different TEF values was obtained with two column combinations, DB-1 x VF-23 and DB-1 x LC-50. When other types of first-dimension columns were used (and orthogonality was partly sacrificed), a DB-XLB column combined with 007-65HT, VF-23 and LC-50 was found to provide a complete separation of all 29 priority congeners. Next, the potential of these three column combinations for real-life analysis was preliminarily studied. With a spiked and fractionated milk extract, DB-XLB x LC-50 was found to be the most powerful column combination, because of the good separation of the 29 priority congeners from each other as well as from the matrix constituents. Quantitative performance (close to three-order linearity; LODs, 30-150 fg injected; R.S.D.s, 1.5-6.5% (n = 10)) was satisfactory.

Cyclin C is a primary 1alpha,25-dihydroxyvitamin D(3) responding gene.

1alpha,25-dihydroxyvitamin D(3) (VD) is a pleiotropic nuclear hormone that also has effects on cell cycle regulation. VD and its synthetic analogues are known inhibitors of cellular growth and inducers of apoptosis, however, the primary mediator genes of these effects largely remain unknown. In order to identify novel targets for VD, that may be involved in the regulation of the cell cycle, a differential display PCR (ddPCR) approach was applied to the MCF-7 human breast cancer cell line, which provided the gene for cyclin C as an interesting candidate. Quantitative assessment of cyclin C expression showed that the gene was significantly upregulated by VD and its analogues, EB1089 and CB1093 both on the level of mRNA expression and more so on the level of protein expression in MCF-7 cells. Upregulation of cyclin C protein expression could also be confirmed in MeWo human melanoma and in U937 human promyelocytic leukemia cells. This observation adds a new gene candidate to the list of primary VD responding genes. Cyclin C is not a typical cyclin, as it apparently modulates the activity of the RNA polymerase II complex, which provides fresh insight into the mechanisms of cell cycle and general transcriptional regulation by VD and its analogues.

Potentiation by vitamin D analogs of TNFalpha and ceramide-induced apoptosis in MCF-7 cells is associated with activation of cytosolic phospholipase A2.

Synthetic analogs of vitamin D induce apoptosis in cultured breast cancer cells and cause regression of experimentally-induced rat mammary tumors. To further elucidate the mechanisms involved, we have examined interactions between two vitamin D analogs (CB1093 and EB1089) and known mediators of apoptosis, TNFalpha and ceramide. Pretreatment of MCF-7 breast cancer cells with CB1093 and EB1089 substantially potentiated cytotoxic effects of TNFalpha as assessed by cell viability assay, DNA fragmentation and videomicroscopy. No significant changes in the levels of TNFalpha or TNF-RI transcripts were detected. CB1093 primed cells demonstrated enhanced responsiveness to cell permeable C2-ceramide in terms of increased DNA fragmentation and loss of cell viability. Activation of cytosolic phospholipase A2 (cPLA2) has been implicated in TNFalpha-mediated apoptosis. As assessed by [3H]-arachidonic acid release, cells primed for 48 h with CB1093 (50 nM) showed enhanced cPLA2 activation in response to TNFalpha or ceramide. CB1093 treatment alone led to cPLA2 activation and loss of cell viability which was inhibited by the specific inhibitor AACOCF3. These results suggest that TNFalpha and vitamin D analogs share a common pathway leading to apoptosis involving cPLA2 activation and/or ceramide generation.

Positive and negative interaction of 1,25-dihydroxyvitamin D3 and the retinoid CD437 in the induction of human melanoma cell apoptosis.

The natural ligands of the nuclear receptors vitamin D receptor (VDR) and retinoic acid receptor (RAR), i.e., 1alpha,25-dihydroxyvitamin D3 (VD) and all-trans retinoic acid, have important effects on the proliferation, differentiation and apoptosis of a variety of malignant cells, including melanoma. The therapeutic potential of the 2 nuclear hormones can be enhanced by the use of synthetic analogues. In this study, the 2 human melanoma cell lines WM1341 and MeWo were compared for the combined effect of VD and synthetic retinoids. Both cell lines expressed reasonable amounts of VDR, RARgamma and retinoid X receptor alpha and differed only in the relative expression of RARalpha and beta. From 9 functional variants of retinoids, only the RARgamma-selective retinoid CD437 showed, in both cell lines, a significant anti-proliferative effect. In MeWo cells, but not in WM1341 cells, VD induced growth arrest but showed no synergistic interaction with the effects of CD437. In contrast, VD induced apoptosis in WM1341, but not in MeWo, cells. CD437 was a strong inducer of apoptosis in both melanoma cell lines. Parallel treatment with CD437 and VD resulted in synergistic enhancement of apoptosis in WM1341 cells, whereas a clear decrease in induction of apoptosis in MeWo cells occurred. Our results indicate that a combined treatment of melanoma with VD and selected retinoids is promising but should be adapted to individual types of tumor.

Differential apoptotic response of human melanoma cells to 1 alpha,25-dihydroxyvitamin D3 and its analogues.

Pleiotropic actions of the biologically active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (VD), include antiproliferative effects in both normal human melanocytes and malignant melanoma cell lines. In this study the actions of VD and its low calcemic analogues EB1089 and CB1093, have been examined in two human melanoma cell lines MeWo and WM1341. Both cell lines express similar amounts of vitamin D receptor mRNA and show functional gene regulatory effects in response to VD and its analogues. VD, EB1089 and CB1093 induced apoptosis only in WM1341 cells and not in MeWo cells, even though both cell lines responded well to etoposide, a strong inducer of apoptosis. Additionally, these results were confirmed by analysis of cell morphology. Interestingly in WM1341 cells, CB1093 was found to be more potent in inducing apoptosis than EB1089 and the natural hormone. Moreover, CB1093 appeared to induce apoptosis at a relatively low concentration of 0.1 nM, whereas greater than tenfold higher concentrations of VD and EB1089 were needed to obtain comparable effects. These observations highlight CB1093 as a promising drug for a future treatment against specific types of melanoma.

Sensitive induction of apoptosis in breast cancer cells by a novel 1,25-dihydroxyvitamin D3 analogue shows relation to promoter selectivity.

The biologically active form of vitamin D3, the nuclear hormone 1 alpha,25-dihydroxyvitamin D3 (VD), is an important regulator of cellular growth, differentiation, and death. The hormone mediates its action through the activation of the transcription factor VDR, which is a member of the superfamily of nuclear receptors. In most cases the ligand-activated VDR is found in complex with the retinoid X receptor (RXR) and stimulates gene transcription mainly from VD response elements (VDREs) that are formed by two hexameric core binding motifs and are arranged either as a direct repeat spaced by three nucleotides (DR3) or as an inverted palindrome spaced by nine nucleotides (1P9). The two VD analogues CB1093 and EB1089 are both very potent inhibitors of the proliferation of MCF-7 cultured breast cancer cells displaying approximately 100-fold lower IC50 values (0.1 nM) than the natural hormone. In addition, CB1093 is even more potent in vivo than EB1089 in producing regression of experimental mammary tumors. Moreover, both VD analogues induce apoptosis in MCF-7 cells, but CB1093 is effective at concentrations approximately 10-fold lower than EB1089. In accordance, the reduction of Bcl-2 protein expression showed CB1093 to be more potent than EB1089. This suggests that the antiproliferative effect of CB1093 may be related mainly to its apoptosis inducing effect, whereas EB1089 may preferentially have effects on growth arrest. EB1089 is known to result in a selectivity for the activation of IP9-type VDREs, whereas CB1093 shows a preference for the activation of DR3-type VDREs. This promoter selectivity suggests that the effects of VD and its analogues on growth arrest and the induction of apoptosis may be mediated by different primary VD responding genes. In conclusion, CB1093 was found to be a potent inhibitor of rat mammary tumor growth in vivo. CB1093 also displayed a high potency in vitro in the induction of apoptosis, a process that may be linked to a promoter selectivity for DR3-type VDREs.

Potent gene regulatory and antiproliferative activities of 20-methyl analogues of 1,25 dihydroxyvitamin D3.

The biological active form of vitamin D3, 1,25-dihydroxyvitamin D3 (VD), regulates cellular growth and differentiation. This provides the hormone with an interesting therapeutic potential. However, hypercalcemia is a side effect, which is caused by VD's classical action, the regulation of calcium homeostasis. This made the need for VD analogues with selectively increased cell regulatory properties. Studies with 20-epi analogues pointed out the importance of the carbon-20 position and led to the development of 20-methyl derivatives of VD. In this report the biological properties of the compounds ZK161422 and ZK157202, which are 20-methyl- and 20-methyl-23-eneanalogues, respectively, have been analyzed in comparison with VD. Both compounds show about 2-fold lower affinity to the VD receptor (VDR) than VD. However, compared to VD, their antiproliferative effect is up to 30-fold higher on human peripheral blood mononuclear cells and even up to 300-fold higher on human breast cancer MCF-7 cells. Whereas the hypercalcemic effect for ZK157202 is also increased 10-fold, ZK161422 has the same calcium-mobilizing potency as VD. Moreover, ZK161422, but not ZK157202, showed preference for gene activation from a promoter carrying a VD response element with a palindromic arrangement of two hexameric receptor binding sites spaced by 9 nucleotides (IP9) rather than for activation from a response element formed by a direct repeat spaced by 3 nucleotides (DR3). This observation supports a model, in which promoter selectivity reflects the selectively increased antiproliferative effect of VD analogues.

The potent anti-proliferative effect of 20-epi analogues of 1,25 dihydroxyvitamin D3 in human breast-cancer MCF-7 cells is related to promoter selectivity.

The hormone 1,25-dihydroxyvitamin D3 (VD) has a potential use as an anti-tumor agent, but its clinical applications are restricted by its strong calcemic activity. This has led to the development of VD analogues with selectively increased growth-inhibitory activity. One of the most potent analogues is KH1060, which is a 20-epi-22-oxa-derivative of VD. In human breast cancer MCF-7 cells, we studied the growth-inhibitory activities of a set of 8 analogues that cover conservative structural changes from 20-epi-VD (MC1288) to KH1060. In the same cellular system, we analyzed the potential of these 8 analogues to stimulate reporter gene activity driven by a recently discovered novel-type VD response element. We found that this VD response element is more appropriate than classical VD response elements to correlate anti-proliferative effects of VD analogues with their gene-regulatory properties.

Identification of natural monomeric response elements of the nuclear receptor RZR/ROR. They also bind COUP-TF homodimers.

The receptor RZR/ROR is an important member of the nuclear receptor superfamily and has recently been shown to be the nuclear receptor for the pineal gland hormone melatonin. RZR/ROR binds as a monomer to DNA, and the human 5-lipoxygenase gene has been identified as the first RZR/ROR/melatonin-responding gene. Another prominent nuclear receptor is COUP-TF, which binds as a dimer to DNA. In this study, the sequences of known promoter regions of genes that may be involved in the physiological action of melatonin have been screened for putative monomeric RZR/ROR response elements. The binding of RZR/ROR and COUP-TF was compared and quantified on a set of 12 putative response elements. Interestingly, COUP-TF homodimers were found to bind with high affinity to some of the monomeric RZR/ROR response elements. Four RZR/ROR response elements, found in the genes of the mouse bifunctional enzyme, the rat bone sialoprotein, mouse Purkinje cell protein 2, and human p21(WAF1/CIP1), were shown to be inducible by melatonin under conditions of low constitutive activity. Surprisingly, the constitutive activity of COUP-TF was also stimulated by an unknown serum compound. The novel Purkinje cell protein 2 and p21(WAF1/CIP1) RZR/ROR/melatonin-responding genes may be the key for understanding the role of RZR/RORalpha in the mouse mutation staggerer and the antiproliferative action of melatonin, respectively.

Centrifuge man-rating of a conceptual internal abdominal bladder restraint in an extended coverage anti-G suit.

An extended coverage anti-G suit, has been demonstrated to improve +Gz tolerance substantially. In some pilots/subjects, however, the abdominal bladder of the anti-G suit may expand excessively upward and inward causing discomfort and pain. This man-rating was performed to evaluate the effects on +Gz protection of an internal abdominal bladder restraint in the Swedish Tactical Flight Combat Suit (TFCS) used in conjunction with pressure breathing during G (PBG). The tests were executed in the Armstrong Laboratory Centrifuge at Brooks AFB with four Swedish test fighter pilots. The centrifuge profiles included gradual onset runs (GOR, relaxed) and rapid onset runs (ROR, with straining), as well as simulated aerial combat maneuver (SACM) runs up to +9 Gz until subjects experienced light loss or fatigue or surpassed 228 s. All subjects withstood 60 s at +9 Gz during GOR and ROR runs with and without abdominal bladder restraint. No difference There was no difference in SACM duration times. In three of four subjects, abdominal pain or discomfort experienced without abdominal bladder restraint disappeared with the addition of a bladder restraint. Ratings of perceived exertion (after 5 peaks at +9 Gz in the SACM), subjective +Gz tolerance, overall comfort, fatigue, and heat stress demonstrated no relevant differences with and without abdominal bladder restraint. Therefore, to enhance comfort, it seems possible to modify the TFCS by adding an abdominal bladder internal restraint without compromising its operational +Gz protection.

The anti-proliferative effect of vitamin D3 analogues is not mediated by inhibition of the AP-1 pathway, but may be related to promoter selectivity.

The hormone 1,25-dihydroxyvitamin D3 (VD) is able to induce cellular differentiation and to inhibit cellular proliferation, which provides it with an interesting therapeutic potential in cancer. However, side effects of VD on homeostasis (eg hypercalcemia) had made the need for the development of VD analogues with low calcemic effect. On the human breast cancer cell line MCF-7 we obtained with the VD analogue EB1089 an about 100-fold higher anti-proliferative effect than with VD. We found that this difference in biological activity is neither related to increased functional affinity to the VD receptor nor to repression of AP-1 activity. The physiologically most prominent complex of the VD receptor is a heterodimer with the retinoid X receptor that binds VD response elements formed two hexameric core binding motifs being arranged either as direct repeats spaced by 3 nucleotides (DR3s) or as inverted palindromes spaced by 9 nucleotides (IP9s). We observed that EB1089 stimulates transcriptional activation from IP9-type elements at clearly lower concentrations than from DR3-type elements. It is possible that IP9-type response elements play an important role in or contribute to the control of cell proliferation, so that promoter-selectivity may explain the high anti-proliferative effect of EB1089.

The nuclear receptor for melatonin represses 5-lipoxygenase gene expression in human B lymphocytes.

The two subtypes of retinoid Z receptor (RZR alpha and beta) and the three splicing variants of retinoid orphan receptor (ROR alpha 1, alpha 2, and alpha 3) form a subfamily within the superfamily of nuclear hormone receptors. Very recently we found that the pineal gland hormone melatonin is a natural ligand of RZR alpha and RZR beta. Ligand-induced transcriptional control is therefore proposed to mediate physiological functions of melatonin in the brain where RZR beta is expressed, but also in peripheral tissues, where RZR alpha was found. However, no natural RZR responding genes have been identified yet. Here, we report that a response element in the promoter of 5-lipoxygenase binds specifically RZR alpha and ROR alpha 1, but not ROR alpha 2 and alpha 3. 5-Lipoxygenase is a key enzyme in the biosynthesis of leukotrienes, which are known to be allergic and inflammatory mediators. We could show that the activity of the whole 5-lipoxygenase promoter as well as of the RZR response element fused to the heterologous thymidine kinase promoter could be repressed by melatonin. The hormone down-regulated the expression of 5-lipoxygenase about 5-fold in B lymphocytes, which express RZR alpha. In contrast, 5-lipoxygenase mRNA levels were not affected in differentiated monocytic and granulocytic cell lines, which do not express RZR alpha. This indicates that 5-lipoxygenase is the first natural RZR alpha responding gene. Furthermore, our results open up a new perspective in understanding the involvement of melatonin in inflammatory and immunological reactions.

Soluble complement receptor type 1 (CD35) is released from leukocytes by surface cleavage.

The soluble form of complement receptor type 1 in human plasma (sCR1) might correspond to the shedding of the receptor by proteolytic cleavage at the cell surface. A new enzyme-linked immunosorbent assay (ELISA) was established to specifically measure membrane-bound CR1 using a rabbit polyclonal antibody against a 19-amino acid peptide corresponding to the C-terminal sequence of the intracellular domain of CR1 (mCR1-ELISA). This ELISA measured CR1 from solubilized erythrocyte membranes, polymorphonuclear leukocytes (PMN), a B lymphocyte cell line and renal podocyte-derived urinary vesicles in a dose-dependent manner. In contrast, and similarly to recombinant soluble CR1 which lacks the intracellular domain of CR1, plasmatic sCR1 was not recognized, suggesting that sCR1 corresponds to an extracellular fragment of whole CR1. In vitro, PMN were shown to release a soluble form of CR1 which was also not recognized in the mCR1-ELISA, and whose size was smaller (5 kDa) than the CR1 of PMN cell membranes. The release of soluble CR1 was highest for PMN and HL60 cells, followed by U937 cells and three different B lymphocyte cell lines, whereas T lymphocyte cell lines did not release soluble CR1. The levels of CR1 gene expression were also higher in PMN compared to remaining blood leukocytes and the different cell lines tested above. Incubation of PMN with formyl-methionyl-leucyl-phenylalanine, tumor necrosis factor-alpha or lipopolysaccharide accelerated the release of soluble CR1, and incubation with granulocyte/macrophage colony-stimulating factor resulted in sustained CR1 gene expression and higher total soluble CR1 release. Our results suggest that soluble CR1 is produced by cleavage of cell surface CR1, and that a large fraction of human plasma sCR1 is cleaved from PMN. The release of sCR1 by leukocytes may play a role in the control of complement activation at sites of inflammation.

Proteolytic cleavage of CR1 on human erythrocytes in vivo: evidence for enhanced cleavage in AIDS.

The number of complement receptor type 1 (CR1; CD35) on human erythrocytes (E) decreases during normal in vivo aging. Patients with acquired immunodeficiency syndrome (AIDS) have an acquired deficiency of CR1 on E. The possible mechanisms responsible for the loss of CR1 from E include the release of small vesicles from the E membrane and proteolytic cleavage of CR1. When compared to E of normal donors and of asymptomatic human immunodeficiency virus HIV+ subjects, E of patients with AIDS had fewer CR1/E (p < 0.001), but had the same number of two glycosylphosphatidylinositol-anchored proteins, decay-accelerating-factor (DAF) and CD59. When compared to young E, old E separated by density gradients on Percoll had fewer CR1 [six normal subjects, mean loss: 50.4 +/- 4.9 (SEM) %], DAF (34.4 +/- 1.2%) and CD59 (34.5 +/- 2.7%). The loss of CR1 was significantly higher than the loss of DAF and CD59 (p < 0.02). In vitro, ATP depletion of E is responsible for the release of vesicles from the E surface, a reaction that has been called in vitro aging. CR1, DAF and CD59 were lost on ATP-depleted E; however, the loss of CR1 and DAF were identical (six experiments, mean loss of CR1: 28.7 +/- 2.7%, DAF: 26.3 +/- 4.6% and CD59: 20.5 +/- 4%). Thus, the release of vesicles from E cannot explain the specific loss of CR1 in patients with AIDS and would explain only incompletely the loss of CR1 during in vivo aging. In vitro experiments indicated that CR1 was more sensitive to trypsin and papain cleavage than DAF and CD59. Enhanced chemiluminescence Western blotting, using a monoclonal antibody (E11) recognizing fragments of CR1 down to 43 kDa on E exposed to trypsin or papain, indicated that normal E bear fragments of CR1, which are not found on polymorphonuclear leukocytes or on CR1-bearing vesicles in urine. The relative amount of these fragments was increased in patients with AIDS. Taken together these data suggest that the specific loss of CR1 on E in AIDS is due to proteolytic cleavage. The loss of CR1 during in vivo aging also involves proteolytic cleavage, although part of the loss might be explained by other mechanisms including the release of vesicles by E.

Circulating soluble CR1 (CD35). Serum levels in diseases and evidence for its release by human leukocytes.

C receptor type 1 (CR1, CD35) is present in a soluble form in plasma (sCR1). Soluble CR1 was measured with a specific ELISA assay in normal individuals and in patients with different diseases. The mean serum concentration of sCR1 in 31 normal donors was 31.4 +/- 7.8 ng/ml, and was identical in plasma. An increase in sCR1 was observed in 36 patients with end-stage renal failure on dialysis (54.8 +/- 11.7 ng/ml, p < 0.0001), and in 22 patients with liver cirrhosis (158.3 +/- 49.9 ng/ml, p < 0.0001). The mean sCR1 levels dropped from 181 +/- 62.7 to 52.1 +/- 24.0 ng/ml (p < 0.001) in nine patients who underwent liver transplantation, and was 33.5 +/- 7.3 in 10 patients with functioning renal grafts, indicating that the increase in sCR1 was reversible. Soluble CR1 was elevated in some hematologic malignancies (> 47 ng/ml), which included B cell lymphoma (12/19 patients), Hodgkin's lymphoma (4/4), and chronic myeloproliferative syndromes (4/5). By contrast, no increase was observed in acute myeloid or lymphoblastic leukemia (10) or myeloma (5). In two patients with chronic myeloproliferative syndromes, sCR1 decreased rapidly after chemotherapy. The mean concentration of sCR1 was not significantly modified in 181 HIV-infected patients at various stages of the disease (34.8 +/- 14.4 ng/ml), and in 13 patients with active SLE (38.3 +/- 19.6 ng/ml), although in both groups the number of CR1 was diminished on E. There was a weak but significant correlation between sCR1 and CR1 per E in HIV infection and SLE (r = 0.39, p < 0.0001, and r = 0.60, p < 0.03 respectively). In vitro, monocytes, lymphocytes, and neutrophils were found to release sCR1 into culture supernatants. In vivo, sCR1 was detected in the serum of SCID mice populated with human peripheral blood leukocytes. The sCR1 levels correlated with those of human IgG (r = 0.97, p < 0.0001), suggesting synthesis of sCR1 by the transferred lymphocytes. The mechanisms underlining the increased levels of sCR1 and its biologic consequences remain to be defined.