Genotype-phenotype correlation in a large English cohort of patients with autosomal recessive ichthyosis.

BACKGROUND: Recessive forms of congenital ichthyosis encompass a group of rare inherited disorders of keratinization leading to dry, scaly skin. So far, 13 genes have been implicated, but there is a paucity of data on genotype-phenotype correlation in some populations. OBJECTIVES: We compiled an English cohort of 146 individuals with recessive ichthyosis and assessed genotype-phenotype correlation. METHODS: Deep phenotyping was undertaken by history-taking and clinical examination. DNA was screened for mutations using a next-generation sequencing ichthyosis gene panel and Sanger sequencing. RESULTS: Cases were recruited from 13 National Health Service sites in England, with 65% of patients aged < 16 years at enrolment. Pathogenic biallelic mutations were found in 83% of cases, with the candidate gene spread as follows: TGM1 29%, NIPAL4 12%, ABCA12 12%, ALOX12B 9%, ALOXE3 7%, SLC27A4 5%, CERS3 3%, CYP4F22 3%, PNPLA1 2%, SDR9C7 1%. Clinically, a new sign, an anteriorly overfolded ear at birth, was noted in 43% of patients with ALOX12B mutations. The need for intensive care stay (P = 0·004), and hand deformities (P < 0·001), were associated with ABCA12 mutations. Self-improving collodion ichthyosis occurred in 8% of the cases (mostly TGM1 and ALOX12B mutations) but could not be predicted precisely from neonatal phenotype or genotype. CONCLUSIONS: These data refine genotype-phenotype correlation for recessive forms of ichthyosis in England, demonstrating the spectrum of disease features and comorbidities, as well as the gene pathologies therein. Collectively, the data from these patients provide a valuable resource for further clinical assessment, improving clinical care and the possibility of future stratified management. What's already known about this topic? Recessive forms of ichthyosis are rare but often difficult to diagnose. Mutations in 13 genes are known to cause recessive forms of ichthyosis: ABCA12, ALOX12B, ALOXE3, CERS3, CYP4F22, LIPN, NIPAL4, PNPLA1, SDR9C7, SLC27A4, SULT2B1, ST14 and TGM1. Some phenotypic features may associate with certain gene mutations, but paradigms for genotype-phenotype correlation need refining. What does this study add? The genotypic spectrum of recessive ichthyosis in England (based on 146 cases) comprises TGM1 (29%), NIPAL4 (12%), ABCA12 (12%), ALOX12B (9%), ALOXE3 (7%), SLC27A4 (5%), CERS3 (3%), CYP4F22 (3%), PNPLA1 (2%) and SDR9C7 (1%). New or particular phenotypic clues were defined for mutations in ALOX12B, ABCA12, CYP4F22, NIPAL4, SDR9C7 and TGM1, either in neonates or in later life, which allow for greater diagnostic precision. In around 17% of cases, the molecular basis of recessive ichthyosis remains unknown.

The role of receptor MAS in microglia-driven retinal vascular development.

OBJECTIVE: The receptor MAS, encoded by Mas1, is expressed in microglia and its activation has been linked to anti-inflammatory actions. However, microglia are involved in several different processes in the central nervous system, including the promotion of angiogenesis. We therefore hypothesized that the receptor MAS also plays a role in angiogenesis via microglia. APPROACH AND RESULTS: To assess the role of MAS on vascular network development, flat-mounted retinas from 3-day-old wild-type (WT) and Mas1-/- mice were subjected to Isolectin B4 staining. The progression of the vascular front was reduced (- 24%, p < 0.0001) and vascular density decreased (- 38%, p < 0.001) in Mas1-/- compared to WT mice with no change in the junction density. The number of filopodia and filopodia bursts were decreased in Mas1-/- mice at the vascular front (- 21%, p < 0.05; - 29%, p < 0.0001, respectively). This was associated with a decreased number of vascular loops and decreased microglial density at the vascular front in Mas1-/- mice (-32%, p < 0.001; - 26%, p < 0.05, respectively). As the front of the developing vasculature is characterized by reduced oxygen levels, we determined the expression of Mas1 following hypoxia in primary microglia from 3-day-old WT mice. Hypoxia induced a 14-fold increase of Mas1 mRNA expression (p < 0.01). Moreover, stimulation of primary microglia with a MAS agonist induced expression of Notch1 (+ 57%, p < 0.05), Dll4 (+ 220%, p < 0.001) and Jag1 (+ 137%, p < 0.001), genes previously described to mediate microglia/endothelial cell interaction during angiogenesis. CONCLUSIONS: Our study demonstrates that the activation of MAS is important for microglia recruitment and vascular growth in the developing retina.

Uptake of risk-reducing salpingo-oophorectomy in women carrying a BRCA1 or BRCA2 mutation: evidence for lower uptake in women affected by breast cancer and older women.

BACKGROUND: Bilateral risk-reducing salpingo-oophorectomy (BRRSO) is the only effective way of reducing mortality from ovarian cancer. This study investigates uptake of BRRSO in 700 BRCA1/2 mutation carriers from Greater Manchester. METHODS: Dates of last follow-up and BRRSO were obtained, and the following variables were investigated: ovarian cancer risk/gene, age and breast cancer history. The date of the genetic mutation report was the initiation for Kaplan-Meier analysis. RESULTS: The uptake of BRRSO in BRCA1 mutation carriers was 54.5% (standard error 3.6%) at 5 years post testing compared with 45.5% (standard error 3.2%) in BRCA2 mutation carriers (P=0.045). The 40-59 years category showed the greatest uptake for BRRSO and uptake was significantly lower in the over 60 s (P<0.0001). Of the unaffected BRCA1 mutation carriers, 65% (standard error 5.1%) opted for surgery at 5 years post-testing compared with 41.1% (standard error 5.1%) in affected BRCA1 mutation carriers (P=0.045). CONCLUSION: The uptake of BRRSO is lower in women previously affected by breast cancer and in older women. As there is no efficient method for early detection of ovarian cancer, uptake should ideally be greater. Counselling should be offered to ensure BRCA1/2 mutation carriers make an informed decision about managing their ovarian cancer risk.

HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine.

Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-kappaB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1beta, IL-6 and TNF-alpha; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-alpha but not IL-1beta or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.

Heterogeneity of helper/inducer T lymphocytes. I. Lymphokine production and lymphokine responsiveness.

Antigen-specific, Ia-restricted helper/inducer T lymphocytes consist of subsets that can be distinguished by lymphokine secretion. One, called Th1, secretes IL-2 and the other, termed Th2, produces BSF-1/IL-4 in response to stimulation by lectin or antigen receptor signals, and each uses the respective lymphokine as its autocrine growth factor. Cloned lines representing Th2 cells proliferate in response to both IL-2 and their autocrine lymphokine, BSF-1/IL-4, but this proliferation is dependent on the synergistic costimulator activity of the monokine, IL-1. In contrast, Th1 clones proliferate only in response to IL-2, are unresponsive to BSF-1/IL-4, and their growth is unaffected by IL-1. These response patterns are not attributable to variations in culture conditions but apparently reflect intrinsic properties of the two T cell subsets. Moreover, the unresponsiveness of Th1 cells to BSF-1/IL-4 may be related to lower levels of expression of surface receptors for this lymphokine. These results may explain the observed heterogeneity among bulk populations of T cells in terms of lymphokine responsiveness and requirement for accessory factors (costimulators). In addition, our findings suggest that IL-2, unlike BSF-1/IL-4, is a fully competent growth factor that is potentially involved in antigen-independent expansion of bystander T cells present at sites of immune stimulation.

Addition of pathology and biomarker information significantly improves the performance of the Manchester scoring system for BRCA1 and BRCA2 testing.

BACKGROUND: Selection for genetic testing of BRCA1/BRCA2 is an important area of healthcare. Although testing costs for mutational analysis are falling, costs in North America remain in excess of US$3000 (UK price can be 690 pounds). Guidelines in most countries use a 10-20% threshold of detecting a mutation in BRCA1/2 combined within a family before mutational analysis is considered. A number of computer-based models have been developed. However, use of these models can be time consuming and difficult. The Manchester scoring system was developed in 2003 to simplify the selection process without losing accuracy. METHODS: In order to increase accuracy of prediction, breast pathology of the index case was incorporated into the Manchester scoring system based on 2156 samples from unrelated non-Jewish patients fully tested for BRCA1/2, and the scores were adapted accordingly. Results/ DISCUSSION: Data from breast pathology allowed adjustment of BRCA1 and combined BRCA1/2 scores alone. There was a lack of pathological homogeneity for BRCA2, therefore specific pathological correlates could not be identified. Upward adjustments in BRCA1 mutation prediction scores were made for grade 3 ductal cancers, oestrogen receptor (ER) and triple-negative tumours. Downward adjustments in the score were made for grade 1 tumours, lobular cancer, ductal carcinoma in situ and ER/HER2 positivity. Application of the updated scoring system led to four and nine more mutations in BRCA1 being identified at the 10% and 20% threshold, respectively. Furthermore, 65 and 58 fewer cases met the 10% and 20% threshold, respectively, for testing. Moreover, the adjusted score significantly improved the trade-off between sensitivity and specificity for BRCA1/2 prediction.

Hepatic fibrosis in patients with chronic hepatitis C assessed by transient elastography: implications for determining the efficacy of antiviral therapy.

BACKGROUND: The efficacy of combination therapy with peginterferon plus ribavirin to eradicate viral infection in patients with chronic hepatitis C (CHC) is well established; moreover, it is able to arrest or even reverse liver fibrosis. AIMS: To analyze the measurements of hepatic stiffness as an index of liver fibrosis using transient elastography (TE) in patients who underwent a sustained virological response (SVR) during long-term follow-up; comparing the changes in the severity of fibrosis with non-responders patients. MATERIAL AND METHODS: After hepatic fibrosis was studied in three patients with CHC who underwent a SVR during long-term follow up, a prospective study was initiated in 24 patients with CHC who received combination therapy to compare the evolution of fibrosis in those with SVR and those who were non-responders. The genotype of hepatitis C virus (HCV) and the degree of viremia were determined. METAVIR scoring system was used for liver fibrosis. Hepatic stiffness was measured by TE. RESULTS: Of the initial three patients pre-treatment liver biopsies revealed active disease and fibrosis (stage 3) in two and mild fibrosis (stage 1) in one. After several years of follow up serum AST/ALT levels were normal and HCV RNA was undetectable in each case; in contrast to the baseline histological assessments of fibrosis, values for hepatic stiffness (3.4-6.9 KPa) were compatible with an absence of any appreciable hepatic fibrosis. In the prospective study, 8 patients underwent a SVR and 16 were non-responders. TE indicated that the severity of hepatic fibrosis in the SVR group improved in 7 (88%) patients, whereas in the non-responder it improved in only 4 (25%) (p < 0.05). The difference between development of severe fibrosis (F > or = 3) in responders and non-responders was not significant (p = 0.23), possibly due to the small sample size. CONCLUSIONS: Regression of hepatic fibrosis appears to be common in patients with CHC who undergo a SVR. TE is a simple non-invasive technique that enables multiple assessments of the severity of hepatic fibrosis to be made efficiently during long-term follow-up of patients with CHC who receive combination antiviral therapy.

Meiotic maturation in Xenopus oocytes: a link between the cessation of protein secretion and the polarized disappearance of Golgi apparati.

We have studied the relationship between the timing of the late meiotic events that occur during progesterone-induced oocyte maturation, and intracellular protein transport. We have monitored the secretion of chick oviduct proteins from Xenopus laevis oocytes microinjected with polyadenylated mRNA and found that chick ovalbumin and lysozyme are not secreted during the second meiotic metaphase, in contrast to the earlier prophase stage. Maturation had no detectable effect on the glycosylation of ovalbumin, whereas it affected the glycosylation of chick ovomucoid. As maturation proceeded, the Golgi apparati disappeared in a polarized fashion, beginning in the vegetal half. This disappearance coincided temporally and spatially with that of the nuclear envelope. We speculate that Golgi apparatus disappearance and the block in secretion are causally related.

Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5.

A complementary DNA clone has been isolated that encodes a coxsackievirus and adenovirus receptor (CAR). When transfected with CAR complementary DNA, nonpermissive hamster cells became susceptible to coxsackie B virus attachment and infection. Furthermore, consistent with previous studies demonstrating that adenovirus infection depends on attachment of a viral fiber to the target cell, CAR-transfected hamster cells bound adenovirus in a fiber-dependent fashion and showed a 100-fold increase in susceptibility to virus-mediated gene transfer. Identification of CAR as a receptor for these two unrelated and structurally distinct viral pathogens is important for understanding viral pathogenesis and has implications for therapeutic gene delivery with adenovirus vectors.

Biosynthesis of the human C3b/C4b receptor during differentiation of the HL-60 cell line. Identification and characterization of a precursor molecule.

The C3b receptor (C3bR) of the human promyelocytic leukemia cell line (HL-60) was induced by incubating these cells with dimethylsulfoxide (DMSO) or retinoic acid. A majority of differentiated (DMSO- or retinoic acid-treated) but not undifferentiated cells formed rosettes with C3b-coated erythrocytes and were morphologically mature granulocytes. HL-60 cells were surface- or biosynthetically labeled and then solubilized in 1% Nonidet P-40 in the presence of multiple protease inhibitors. The C3bR was isolated either by immunoprecipitation with anti-C3bR antibodies or by affinity chromatography with hemolytically inactive components in which the internal thioester bond within the alpha-chain was cleaved (iC3)- or iC4-Sepharose. Autoradiographs of NaDodSO4-polyacrylamide gels indicated that the surface-labeled C3bR on the differentiated cells had an Mr of 210,000 (nonreduced) or 240,000 (reducing conditions). The bulk (approximately 85%) of the radiolabeled material that was isolated from biosynthetically labeled cells co-migrated with the surface-labeled band. A small fraction (approximately 15%) of the biosynthetically labeled material that was isolated by affinity chromatography or immunoprecipitation had an Mr of 188,000, which did not correspond to any surface-labeled band. This putative precursor molecule was characterized by pulse-chase experiments and by analysis of its carbohydrate. In pulse-chase (15-min pulse) studies of differentiated cells, only the 188,000-mol wt molecule was detected at 0 h. By 2 h, greater than 80% of counts had chased from the 188,000 to the 210,000-mol wt molecule. Treatment of these two molecules with endoglycosidases indicated that the 188,000-mol wt molecule possessed high mannose oligosaccharides, while the mature C3bR had complex oligosaccharides. We conclude from these data that the 188,000-mol wt molecule is a precursor of the C3b receptor of HL-60 cells. Other experiments indicated that the half-maximal time for newly synthesized receptor to attain an Mr of 210,000 was 45 min, and that the t1/2 for the disappearance of the receptor on the surface of differentiated HL-60 cells in tissue culture was approximately 10 h. The ability to observe the induction of the C3b receptor as the HL-60 cell line differentiates is an instructive model system to study the biosynthesis of a human integral membrane receptor glycoprotein.

The mechanism of intestinal uptake and transcellular transport of IgG in the neonatal rat.

The transport of immunoglobulins across the intestinal mucosa of neonatal rats provides an excellent model for the study of transcellular protein transport. The mechanism of intestinal uptake and transcellular transport of plasma proteins has been studied in 12-14-day old rats using intraduodenally administered radioiodinated proteins. Appreciable quantities of rat IgG, mouse IgG, rabbit IgG, and all four subclasses of human IgG were taken up by the intestinal wall (19-54% of administered dose at 4 hr) and transported to the animal (10-35% of administered dose at 4 hr). In contrast there was little or no uptake of human IgM, IgA, and IgE and little or no transport of human IgM, IgA, IgD, IgE, albumin, transferrin, and ceruloplasmin. Both the uptake and transport of labeled IgG were significantly inhibited by unlabeled IgG. Further insight into the transport process was obtained from the observation that an appreciable proportion of the label of IgG in intestinal wall homogenates, but not in plasma or intestinal washings, migrated in a sucrose ultracentrifugation gradient much more rapidly than did the administered 7S molecules. This pattern was not observed with other proteins studied. This apparent binding of labeled IgG was also markedly inhibited by unlabeled IgG. In subcellular fractionation studies of intestinal homogenates the complexed labeled IgG was shown to be associated predominantly with cell membrane rather than cell sap fractions. In addition IgG could be shown to bind to purified enterocyte microvillous membranes in vitro. IT IS CONCLUDED THAT IN THE NEONATAL RAT: (a) the major processes involved in both intestinal uptake and transport of IgG are specific and saturable; (b) intestinal transport is associated with complexing of IgG molecules with membranes, most probably with enterocyte microvillous membranes; and (c) the part of the IgG structure involved in this process is probably similar to that involved in the concentration-catabolism effect but is not identical to that mediating other non-antigen combining functions of IgG. Our data are consistent with the existence of specific receptors for IgG on enterocyte microvillous membranes of the neonatal rat. Such receptors would be necessary for the specific uptake and transport of these molecules.

Augmentation of albumin but not fibrinogen synthesis by corticosteroids in patients with hepatocellular disease.

Simultaneous studies of albumin and fibrinogen metabolism have been conducted using the carbonate-(14)C method before and after a 13 day course of prednisolone in eight patients with hepatocellular disease. Initially six patients were hypoalbuminemic. The mean plasma albumin and fibrinogen concentrations and albumin and fibrinogen synthetic rates were all lower than the corresponding values in a group of control subjects. Prednisolone therapy was associated with significant increases in the plasma concentration and synthetic rate of albumin but changes in the intravascular albumin pools were not significant. It is inferred that a low synthetic rate of albumin in a patient with liver disease does not necessarily represent the maximum capacity of the diseased liver to synthesize this protein. Changes in the plasma concentration, intravascular pool, and synthetic rate of fibrinogen were small and inconsistent. The data are compatible with a selective action of corticosteroids on hepatic protein metabolism and with the existence of different mechanisms for the control of albumin and fibrinogen synthesis.

The measurement of the synthetic rate of bilirubin from hepatic hemes in patients with acute intermittent porphyria.

A new method for the direct measurement in vivo of the synthetic rate of bilirubin from hepatic hemes is proposed. This method depends on the application of the labeled precursor-product relationship to the hepatic pool of porphobilinogen, which is a common precursor of both urinary porphobilinogen and hepatic-synthesized bilirubin. The hepatic pool of porphobilinogen is labeled by means of an intravenous injection of delta-aminolevulinic acid-4-(14)C. The proportion of total bilirubin production which is derived from hepatic hemes is calculated from the ratio of the mean (14)C specific activities of stercobilin and porphobilinogen estimated in pooled specimens of feces and urine, respectively. The method can be most readily applied to patients with acute intermittent porphyria, as the appreciable quantities of prophobilinogen in the urine of these patients greatly facilitate the measurement of porphobilinogen-(14)C specific activity. In three patients with acute intermittent porphyria, values obtained for the synthetic rate of bilirubin from hepatic hemes were 20.7, 15.8, and 13.3% of total bilirubin production.

Modulation of a glycoprotein recognition system on rat hepatic endothelial cells by glucose and diabetes mellitus.

The cellular location and carbohydrate specificities of a glycoprotein recognition system on rat hepatic sinusoidal cells have been determined. Purified preparations of endothelial, Kupffer, and parenchymal cells were prepared by collagenase liver perfusion, centrifugation on Percoll gradients, and centrifugal elutriation. (125)I-labeled agalactoorosomucoid, an N-acetylglucosamine-terminated glycoprotein, was selectively taken up in vitro by endothelial cells. Uptake was shown to be protein dependent, calcium ion dependent, and saturable, and could be described by Michaelis-Menten kinetics (apparent K(m) 0.29 muM; apparent maximum velocity 4.8 pmol/h per 5 x 10(6) cells). Uptake was inhibited not only by N-acetylglucosamine, mannose, and mannan but also by glucose, fructose, and a glucose-albumin conjugate. Inhibition by glucose was competitive over a wide range of concentrations and was almost 100% at a glucose concentration of 56 mM. Fasting and the induction of diabetes mellitus prior to isolation of cells was associated with 60% reductions in the recovery of endothelial cells. Uptake by cells isolated from fasted rats was enhanced (apparent maximum velocity 14.3 pmol/h per 5 x 10(6) cells without change in the apparent K(m)). These observations suggest that fasting is associated with a marked increase in the mean number of glycoprotein receptors per endothelial cell isolated from normal rats. This effect of fasting could be due to upregulation of glycoprotein receptors on endothelial cells or to the selective isolation of a subpopulation of endothelial cells from fasted animals that bears more glycoprotein receptors per cell than does another subpopulation of these cells. In addition, in vivo studies of the fate of intravenously administered (125)I-agalactoorosomucoid indicated that its rate of disappearance from plasma, hepatic accumulation, and catabolism were slower in diabetic than in normal rats. The results suggest that modulation of a carbohydrate-mediated glycoprotein recognition system located on hepatic endothelial cells can be induced by glucose and glucose-conjugated proteins and by fasting and diabetes mellitus. The findings in this study suggest a mechanism for abnormal glycoprotein metabolism in diabetes mellitus.

Suppression of hypothalamic-pituitary-adrenal axis responsiveness to stress in a rat model of acute cholestasis.

Cholestatic patients undergoing surgery have increased mortality and demonstrate clinical features suggestive of adrenal insufficiency. To examine whether cholestasis influences the status of the hypothalamic-pituitary-adrenal axis, we evaluated rats with acute cholestasis caused by bile duct resection (BDR) and sham-operated and unoperated controls. Basal unstressed plasma concentrations of ACTH and corticosterone were similar in BDR and sham-operated and unoperated control rats. However, exposure of BDR rats to saturated ether vapor resulted in significantly less ACTH and corticosterone release in plasma than in the control animals. To understand the mechanism(s) of decreased HPA axis responsiveness to ether stress in cholestasis, we administered corticotropin-releasing factor (CRF) and measured hypothalamic content, mRNA levels and in vitro secretion of CRF and arginine vasopressin (AVP), the two principal secretagogues of ACTH. In BDR animals, ACTH responses to CRF were decreased and hypothalamic content of CRF and CRF mRNA expression in the paraventricular nucleus were decreased by 25 and 37%, respectively. Furthermore, CRF release from hypothalamic explants of BDR rats was 23% less than that of controls. In contrast to CRF, hypothalamic content of AVP was 35% higher, AVP mRNA in the paraventricular nucleus was increased by 6.6-fold, and hypothalamic explant release of AVP was 24% higher in BDR rats than in control animals. Pituitary ACTH contents were similar in BDR and sham resected rats, but higher than unoperated controls. These findings demonstrate that acute cholestasis in the rat is associated with suppression of hypothalamic-pituitary-adrenal axis responsiveness to stress and demonstrate a dissociation between mechanisms of ACTH regulation mediated by CRF and AVP.

Synthesis of antibodies to hepatitis B virus by cultured lymphocytes from chronic hepatitis B surface antigen carriers.

It has been postulated that host immune defects are responsible for the development and persistence of the hepatitis B surface antigen (HBsAg) carrier state. The nature of these defects is unknown, but the absence of a readily detectable antibody response to HBsAg (anti-HBs) may be important. The synthesis of both anti-HBs and antibody to hepatitis B core antigen (anti-HBc) in cultures containing peripheral blood mononuclear cells from chronic HBsAg carriers and from control (antibody-positive) patients was measured in the presence of pokeweed mitogen. Similar amounts of polyclonal IgG and IgM were synthesized by cultures containing lymphocytes from chronic carriers and controls. Anti-HBc was detectable in lymphocyte supernatants from 2 of 20 controls and from 21 of 29 carriers. The presence of anti-HBc synthesis in vitro correlated with high serum titers of anti-HBc. In contrast, anti-HBs was detected in lymphocyte supernatants from 6 of 20 controls (predominantly in those who had high serum titers of anti-HBs) but in none of the supernatants from 29 HBsAg carriers. In order to identify the mechanisms for the lack of detectable anti-HBs synthesis by chronic HBsAg carrier lymphocytes, co-culture experiments were performed using T and B lymphocyte fractions that had been purified by affinity chromatography. B lymphocytes from carriers co-cultured with allogeneic irradiated ("helper") T lymphocytes from controls synthesized normal amounts of IgG, IgM, and anti-HBc but still did not synthesize detectable amounts of anti-HBs. In the converse experiments, B lymphocytes from controls were co-cultured with irradiated T lymphocytes from carriers. The T lymphocytes from 16 of 24 carriers augmented anti-HBs production by control B cells normally, the remaining eight did not. Finally, mixtures of control B cells and control irradiated T lymphocytes were co-cultured with T lymphocytes from chronic HBsAg carriers. 5 of 12 carriers demonstrated active suppression of anti-HBs production, and in three this suppression was specific, as IgG and IgM production remained normal. We conclude that chronic HBsAg carriers have a specific B lymphocyte defect in anti-HBs production. In addition, defects in the function of regulatory T lymphocytes may contribute to the absence of anti-HBs synthesis in some HBsAg carriers.

Receptor specific clearance by the reticuloendothelial system in chronic liver diseases. Demonstration of defective C3b-specific clearance in primary biliary cirrhosis.

An approach to the assessment of reticuloendothelial function that quantitates clearance specifically mediated by membrane receptors for C3b and immunoglobulin (Ig)G has been applied in man. Clearance of isologous erythrocytes coated with IgM or C3b or coated with IgG were examined in patients with primary biliary cirrhosis (PBC), chronic hepatitis, or alcoholic cirrhosis and normal control subjects and compared with the clearance of aggregated human serum albumin. Clearance of these three types of particles varied independently. None of the patients studied had a defect in the clearance of aggregated albumin. No patient with PBC (0:6) had delayed clearance of IgG-coated erythrocytes; one of six patients with chronic hepatitis had delayed clearance of these cells. Indeed, four of six with PBC had increased rates of IgG-mediated clearance. In contrast, six out of six patients with PBC had an unequivocal defect in clearance mediated by C3b receptors. The patients with PBC varied widely in terms of duration of symptoms, degree of cholestasis, and histologic stage of disease. No defect of C3b-mediated erythrocyte clearance was found in the patients with chronic hepatitis or alcoholic cirrhosis. Furthermore, a patient with severe cholestasis secondary to large duct biliary obstruction exhibited normal C3b-mediated clearance. The defect in C3b-mediated clearance in PBC did not correlate with serum levels of individual complement components or inhibitors or with the presence of circulating immune complexes as measured by the Clq precipitation assay. Thus, measurements of receptor specific clearance, but not clearance of aggregated proteins, have revealed a highly specific defect in reticuloendothelial function in PBC.

In vitro cell-mediated cytotoxicity in primary biliary cirrhosis and chronic hepatitis. Dysfunction of spontaneous cell-mediated cytotoxicity in primary biliary cirrhosis.

The in vitro cytotoxic function and target cell specificity of peripheral blood lymphocytes from selected patients with primary biliary cirrhosis and hepatitis B surface antigen-negative chronic hepatitis were investigated using 51Cr-labeled human Chang and EL-4 mouse sarcoma cell targets in assays of spontaneous cell-mediated cytotoxicity (SCMC) and mitogen-induced cellular cytotoxicity (MICC). In addition, antibody-dependent cellular cytotoxicity (ADCC) against Chang cells was assessed. At an effector-to-target cell ration of 100:1, the mean SCMC against Chang cells was much less in patients with primary biliary cirrhosis than that in either the controls (P less than 0.001) or the patients with chronic hepatitis (P less than 0.005) whereas the value for patients with chronic hepatitis did not differ significantly from that of the controls. The mean SCMC against EL-4 mouse sarcoma cells was also less in patients with primary biliary cirrhosis than in controls (P less than 0.005) whereas the value for chronic hepatitis was not significantly different from that of the controls or patients with primary biliary cirrhosis. In contrast, MICC against both targets and ADCC against Chang cells were similar for each group. Comparison of SCMC and MICC against both target cells, measured simultaneously, showed similar cytotoxic potenital against both target cells for each group. Effector cells capable of mediating cytotoxicity in each assay were defined by testing the cytotoxic function of lymphocyte subpopulations isolated from two representative patients with each disease using techniques of immunoabsorbent affinity chromatography and Fc receptor binding to antigen-antibody complexes. In both primary biliary cirrhosis and chronic hepatitis SCMC and ADCC were mediated by a subpopulation of lymphocytes which lack surface immunoglobulin (sIg-) and bear Fc receptors (Fc+). In contrast, MICC was mediated by sIg- cells which lack Fc receptors. Lymphocytes bearing sIg- were not cytotoxic in any assay. These results establish a difference in cytotoxic function in primary biliary cirrhosis and chronic hepatitis by defining the presence of a defect in spontaneous cytotoxic function of sIg-, Fc+ lymphocytes against Chang cells in primary biliary cirrhosis.

Quantitative studies of the delivery of hepatic-synthesized bilirubin to plasma utilizing -aminolevulinic acid-4- 14 C and bilirubin- 3 H in man.

After the simultaneous intravenous administration of unconjugated bilirubin-(3)H and delta-aminolevulinic acid-4-(14)C, the plasma disappearance curves of unconjugated bilirubin-(3)H and the plasma appearance curves of biosynthesized unconjugated bilirubin-(14)C have been defined in seven patients, three of whom had acute intermittent porphyria (AIP). The incorporation of (14)C into plasma unconjugated bilirubin, derived by an analysis which involves deconvolution of the two plasma curves, varied between 13.1 and 23.5% (mean 19.3%) of the injected dose in the nonporphyric patients and between 5.4 and 13.6% (mean 8.3%) of the injected dose in the porphyric patients. In five of the patients, the stercobilin-(14)C specific activity in a pooled specimen of feces was measured, enabling the following further values to be calculated: (a) the total (14)C radioactivity incorporated into bilirubin (21.0 and 25.3% [mean 23.2%] of the injected dose in two of the nonporphyric patients and between 8.5 and 25.3% [mean 14.2%] of the injected dose in the porphyric patients), and (b) the proportion of hepatic synthesized bilirubin delivered directly to plasma in the unconjugated form (between 0.520 and 0.904; mean for nonporphyric patients 0.712; mean for porphyric patients 0.614). The results demonstrate that a large proportion of bilirubin derived from hepatic hemes passes through the plasma in the unconjugated form before conjugation and secretion into bile.

Deficiency of the autologous mixed lymphocyte reaction in patients with primary biliary cirrhosis.

In this study we show that patients with primary biliary cirrhosis (PCB) have a marked deficiency in the ability to generate an autologous mixed lymphocyte reaction (AMLR) but have a normal ability to generate an allogeneic mixed lymphocyte reaction (MLR). This deficiency is not due to differences in the time-course of the proliferative response or to an altered response to variable numbers of stimulator cells. The deficiency was consistently found irrespective of the methods used to isolate autologous stimulator cells. Both responder cells and stimulator cells obtained from patients with PBC were similar to normal cells in their ability to generate an MLR in allogeneic normal human serum. In addition, serum from patients with PBC inhibited the ability of normal lymphocytes to generate both the AMLR and MLR to a similar degree, suggesting that the defect of the AMLR in PBC is not due to a serum factor. It has been shown that the responder cell population in the AMLR contains a subpopulation of cells that mediate suppression. Therefore, it is possible that the deficiency of the AMLR may be related to previously described abnormalities of suppressor function in patients with PBC.