Radioimmunoassay for antibodies to cytoplasmic ribosomes in human serum.

A radioimmunoassay for the detection of antibodies in human serum to tritium-labeled HeLa cell cytoplasmic ribosomes was developed with the use of Macaloid for the inhibition of endogenous ribonuclease activity. Antibodies were observed in the serum of patients with systemic lupus erythematosus in high incidence and titer. Patients with rheumatoid arthritis and chronic active hepatitis manifested a lower incidence and titer of antibodies to ribosomes, whereas serums from normal individuals and from patients with sarcoidosis, chronic glomerulonephritis, and malignant tumors showed no significant reactivity with cytoplasmic ribosomes. Maximum inhibition of the reaction was achieved with unlabeled HeLa cell ribosomes or rat liver ribosomes and partial inhibition by purified ribosomal RNA.

Influence of methimazole on murine thyroiditis. Evidence for immunosuppression in vivo.

Thionamide drugs are immunosuppressives in vitro. To examine this action in vivo, A/J mice were immunized with human thyroglobulin (hTg) (0.5 mg intraperitoneal injections for 5 d) beginning on days 6, 24, and 43 with or without methimazole (M) (0.05%) and l-thyroxine (T4) (0.1 micrograms/ml to prevent thyroid hypertrophy) in their water supply. Groups (n = 8) were killed on days 37, 42, and 59. Spontaneous splenic IgG-secreting cells determined by Staphylococcus protein A-linked sheep erythrocytes (SRBC) via indirect plaque-forming cell (PFC) assay indicated polyclonal stimulation induced by the hTg exposure (controls = 2,285 +/- 599, hTg-only = 5,570 +/- 470 PFC per 10(6) spleen cells), but this was significantly reduced in the M plus T4-treated group (3,640 +/- 415 PFC, P = 0.05). hTg antibody was measured by specific PFC assay using hTg-linked SRBC. Anti-hTg PFC were absent in controls and were 147 +/- 41, 25 +/- 8, and 173 +/- 58 PFC per 10(6) spleen cells in the hTg-only groups on days 37, 42, and 59, respectively. Anti-hTg PFC results in the M plus T4-treated animals were significantly reduced to 0, 15 +/- 5, and 63 +/- 30 anti-hTg PFC. Histological examination revealed a marked thyroiditis in hTg-only animals and a significantly reduced degree of mononuclear cell infiltration and follicular destruction in the M plus T4-treated groups (graded 1.9 compared with 3.6 in hTg-only P = less than 0.01). Examination of IgG deposition using fluorescent anti-mouse IgG revealed a similar granular pattern and degree of staining in both immunized groups. Control animals that received concurrent T4 administration alone showed similar hTg-induced murine thyroiditis to non-T4-treated animals and could not explain the apparent immunosuppression observed. In conclusion, these data demonstrated that M reduced both the splenic immune response and the degree of thyroiditis after heterologous Tg immunization, while a quantitative difference in the circulating and intrathyroidally deposited Tg antibody was not detected.

Physicochemical and immunohistological studies of a sulfobromophthalein- and bilirubin-binding protein from rat liver plasma membranes.

Affinity chromatography over bilirubinagarose and sulfobromophthalein (BSP)-agarose was used to isolate two proteins, with high affinities for bilirubin and BSP, respectively, from Triton X-100-solubilized rat liver plasma membranes. The protein eluted from either affinity column migrated as a single band of approximately 55,000 D on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and either protein cochromatographed with both [14C]bilirubin and [35S]BSP on Sephadex G-75. On gradient gels without reduction or SDS, or on Sephadex G-150, the native BSP-binding protein had an estimated molecular mass of approximately 100,000 D. After incubation with SDS, an additional Sephadex G-150 peak of molecular mass of 56,000 D was observed. Both, the 100,000- and 56,000-D G-150 peaks cochromatographed with [35S]BSP. The native protein had an isoelectric point of 3.5, stained with periodic acid-Schiff but not Sudan black, and contained 4 mol of sialic acid per mol of protein. A rabbit antibody to the BSP-binding protein gave a line of identity with both the BSP- and bilirubin-binding antigens, and inhibited the binding of [14C]bilirubin and [35S]BSP, but not [14C]oleate or [14C]taurocholate, to rat liver plasma membranes. Immunohistochemical studies revealed the presence of the antigen on all surface domains of rat hepatocytes, but not on other cell populations from normal rat liver. It was not found in other organs. These data are compatible with the hypothesis that a specific liver cell plasma membrane protein mediates the hepatocytic sequestration of bilirubin and BSP.

Tumorigenicity in nude mice of a human hepatoma cell line containing hepatitis B virus DNA.

The human hepatocellular carcinoma cell line PLC/PRF/5, which synthesizes and secretes hepatitis B surface antigen, was grown under optimal conditions in tissue culture, using Eagle's minimal essential medium supplemented with 10% fetal bovine serum and 10(-11) M triiodothyronine on collagen rafts. Injection s.c. of the PLC/PRF/5 cell line into athymic BALB/c nude mice resulted in the growth of a well-circumscribed, moderately differentiated hepatocellular carcinoma. The intervals until tumor appearance and tumor "take" rates were dependent on inoculum dose. Four to 5 x 10(6) cells induced tumor growth in 29% of 14 injected mice within 29 to 40 days, while 7 to 13 X 10(6) cells induced tumors in all 15 mice within 10 to 12 days after inoculation. Hepatitis B surface antigen was detected in the nude mouse serum and tumor tissue, and its concentration roughly correlated with tumor weight. A low level of antibody against hepatitis B surface antigen was detected in five tumor-bearing animals, as well as in one mouse which did not produce a tumor. Hepatitis B core antigen and its antibody and hepatitis B e antigen and its antibody were not detected in 26 mice, using immunohistochemical and radioimmunoassay methods. alpha-Fetoprotein, carcinoembryonic antigen, and alpha-antitrypsin were detected in nude mice tumors, using the peroxidase-antiperoxidase technique. Finally, hepatitis B virus DNA, identified in the nude mouse tumor by molecular hybridization techniques, was compared to PLC/PRF/5 cell line hepatitis B virus DNA.

Pathology of hepatitis C.

The basic morphological patterns of acute or chronic viral hepatitides are very similar, irrespective of the causative hepatitis viruses A, B, C, D or E. In addition, however, acute and chronic hepatitis C shows characteristic, although not pathognomonic histological changes. These consist of lymphoid aggregates in portal tracts, sometimes with germinal centers, damage of bile duct epithelium, and micro- or macrovesicular steatosis of hepatocytes. A combination of two of these three characteristic alterations is seen in over half of the patients with chronic hepatitis C and is helpful in the histological diagnosis of the disease.

Massive hepatic necrosis after chemotherapy withdrawal in a hepatitis B virus carrier.

A chronic hepatitis B virus (HBV) carrier with diffuse nodular transformation of the liver and malignant lymphoma in the lymph nodes and spleen developed massive hepatic necrosis and died three weeks after the third cycle of chemotherapy. Immunosuppressive drug treatment may favor replication of HBV, resulting in massive hepatocyte destruction when the immune response recovers following withdrawal of chemotherapy. This outcome must be considered in patients with chronic hepatitis B who are treated with a course of prednisone followed by antiviral therapy as well as in HBV carriers following chemotherapy for malignant disease.

Liver-directed gene transfer in non-human primates.

To develop a primate model for liver-directed gene therapy, we studied several gene transfer vehicles and routes in eight rhesus monkeys (Macaca mulatta). For this purpose, we used first-generation, replication-deficient adenoviral vectors carrying the Escherichia coli lacZ gene (Ad.CMVlacZ) or a lacZ-containing plasmid (pCMV beta) with lipofectamine for transfection. The reporter gene construct was infused into either the portal vasculature, common bile duct, or saphenous vein. Adenovirus-mediated gene transfer via the portal vein resulted in expression of lacZ in over 70% of hepatocytes by days 3-7, but was accompanied by acute hepatitis. Adenovirus-mediated gene transfer via the common bile duct resulted in lacZ expression in less than 10% of hepatocytes and was accompanied by portal inflammation. The animals mounted a significant immune response, as demonstrated by adenoviral antigen-induced T-cell proliferation and production of neutralizing anti-adenovirus antibodies and antibodies to E. coli beta-galactosidase (beta-Gal). Activation of the immune response was associated with rapid decrease of the reporter gene by days 13-21. Lipofectamine-mediated gene transfer was inefficient, and no lacZ expression in the liver was detected. To limit the host immune response, 4 animals were immunosuppressed by cyclophosphamide/prednisone and then infused with the Ad.CMVlacZ via the portal vein or the saphenous vein. The monkeys showed sustained expression of lacZ for up to 35 days with no evidence of inflammation. The primates transduced via the saphenous vein showed a level of beta-Gal expression in the liver similar to that of the portal vein-infused animals. In conclusion, adenovirus-mediated gene transfer to non-human primate livers via the portal vein or saphenous vein is efficient, but it results in transient expression and is accompanied by an immune response to both vector and transgene products and acute hepatitis, whereas lipofectamine-mediated transfer is inefficient. Manipulation of the host immune response may expand potential applications of adenoviral vectors for liver-directed gene transfer.

Use of transient CD4 lymphocyte depletion to prolong transgene expression of E1-deleted adenoviral vectors.

E1-deleted adenoviral vectors are increasingly being utilized for in vivo gene transfer. The potential use of these vectors is limited by transient expression of the transgene and a markedly reduced rate of transduction following readministration, presumably due to a host immune response to the vector. We hypothesized that CD4+ lymphocytes are necessary to generate an immune response to these vectors and that administration of a depleting anti-CD4 antibody (GK1.5) might prolong transgene expression in vivo. We found that pretreatment of mice with a single injection (transient depletion) or weekly injections of GK1.5 (persistent depletion), markedly prolonged expression of an adenovirus-encoded tumor necrosis factor (TNF) inhibitor or luciferase gene compared to controls. Moreover, mice treated with GK1.5 showed no antiadenoviral antibody response to repeat administration of the vector and a second adenoviral transgene could be expressed in these animals. However, control mice developed a significant neutralizing antibody response that prevented transgene expression with administration of a second adenovirus. These findings demonstrate that manipulation of the host immune response may expand potential applications of gene transfer utilizing adenoviral vectors.

Immunohistologic analysis of lymphoid cells by a rapid double staining method.

The development of monoclonal antibodies has allowed characterization of subpopulations of lymphoid cells and of their in situ distribution in tissues. The feasibility of simultaneous localization of two surface antigens was studied by double staining with monoclonal antibodies to B cells, T cell subsets and follicular dendritic reticulum cells (DRC) using the avidin-biotin-complex (ABC) method in sections of human lymphoid tissues (tonsil, lymph node, spleen) and a non-lymphoid tissue, endometrium. Color mixture was avoided when an additional incubation with avidin-biotin-labeled peroxidase and subsequent development in the respective substrate of the first sequence preceded the second staining sequence using the primary antibodies at optimal concentrations. The antigenic profiles were portrayed by contrasting and distinct colors of the reaction products. It was observed that T lymphocytes of the cytotoxic/suppressor and helper/inducer phenotypes were topographically associated with each other and with B cells in B and T cell areas of lymphoid tissues as well as in lymphocytic aggregates in endometria. Subpopulations of these cells were mantled by processes of DRCs in lymphoid follicles. The findings indicate that the double ABC staining method can be used for simultaneous demonstration of two surface antigens in tissue sections.

Radioimmunometric assay for hepatitis B surface antigen on the surface of infected cells.

It has been difficult to differentiate hepatitis B surface antigen (HBsAg) on the cell surface from intracellular or intramembranous HBsAg that is not exposed to the host immune response. We describe here a radioimmunometric assay for HBsAg on the cell surface using HBsAg producing hepatocellular carcinoma cell lines and fibroblasts transfected with cloned hepatitis B virus DNA as models. The assay is sensitive, specific, simple and takes approximately 1 1/2 h. The procedure may be modified to compare quantitatively cell surface with intracellular HBsAg and to demonstrate other cell associated hepatitis B virus antigens such as HBcAg and HBeAg.

Histology of the liver.

The embryology, gross morphology, and histology of the normal human liver--the single largest organ in the human body--are described. It is emphasized that liver biopsy specimens must be processed with special care in order to obtain optimal sections. Immunohistologic studies of liver tissue have the potential to yield more information than electron microscopy. In surgical and autopsy liver specimens, some histologic alterations may be prominent, but they often have little significance. On the other hand, some morphologic changes, particularly in needle biopsy specimens, are frequently subtle, but may be of diagnostic importance. The pathologist must be familiar with these histologic variations from the normal liver.

Applicability of imprints to immuno-ultrastructural studies of lymphoid tissues.

In order to evaluate the applicability of imprints to immuno-ultrastructural studies of lymphoid tissues, we compared distribution pattern and morphology of B cells, T cells, T-cell subsets, and follicular dendritic reticulum cells (DRC) at the light and ultrastructural level in imprints and sections of tonsils and lymph nodes. The surface antigenic profile of lymphoid cells was revealed with monoclonal antibodies in an avidin-biotin-peroxidase complex (ABC) method. Distribution of lymphoid cells in coherent areas of imprints recapitulated their disposition in sections of respective lymphoid tissues. Preservation of microanatomical relationships and ultrastructure of lymphoid cells in imprints allowed evaluation of associations and fine structural detail of lymphoid cells. Morphologic configurations of B cells in imprints, confined to round aggregates, were similar to fine structural morphology of B cells in mantle zones (MZ) and germinal centers (GC). Processes of DRCs in imprints formed conformations resembling their meshwork within follicles and mantled lymphoid cells. In imprints and sections, lymphocytes of cytotoxic/suppressor phenotype had a large amount of cytoplasm with many organelles. In contrast, cells of helper/inducer phenotype displayed a high nucleocytoplasmic (N/C) ratio and small numbers of organelles. Thus, imprints represent an easy, fast, and reliable method that lends itself to immunoultrastructural studies of lymphoid tissues.

Antibiotic resistance: relationship to persistence of group A streptococci in the upper respiratory tract.

Despite the use of penicillin for more than 40 years in treating GABHS infections, there has been no significant change in the in vitro susceptibility of GABHS to penicillin. Reported failures to eradicate GABHS from the upper respiratory tracts of patients with pharyngitis and the apparent resurgence of serious Group A streptococcal infections and their sequelae probably are not related to the emergence of penicillin resistance. Although erythromycin resistance in GABHS had been a major problem in Japan and continues to be a major problem in Finland, it has not been a problem in this country. The susceptibility of GABHS to the newer macrolide antibiotics appears to be similar to that of erythromycin. Comprehensive, community-wide programs to continuously monitor for erythromycin resistance in GABHS would be difficult to justify. However, because little is known about how erythromycin resistance in GABHS is acquired or spread, it would be reasonable to periodically monitor isolates of GABHS for erythromycin resistance. A substantial proportion of GABHS are currently resistant to tetracyclines and these agents are inappropriate for treating GABHS infections. Although little recent information is available about the susceptibility of GABHS to sulfonamides, these agents have been shown to be ineffective in eradicating GABHS from the upper respiratory tract regardless of the in vitro sensitivities. GABHS have not been shown to be resistant to any of the commonly used oral cephalosporins; however, there is a great deal of variability among these agents in their activity against GABHS. Clindamycin resistance in GABHS has remained unusual. This agent is an alternative for treating GABHS infections due to macrolide-resistant strains in patients who cannot be treated with beta-lactam antibiotics. There is no reason, based on the in vitro susceptibilities of GABHS, to change the current recommendations for treating GABHS infections with penicillin and for using erythromycin for patients who are allergic to penicillin.

Lyme arthritis in children: clinical epidemiology and long-term outcomes.

OBJECTIVE: Although Lyme disease has become a relatively common cause of arthritis among children in areas of the country in which the disease is endemic, little information is available about the clinical epidemiology and long-term outcomes of children with Lyme arthritis. We conducted a long-term follow-up study to determine the clinical epidemiology of Lyme arthritis in children as well as their long-term outcomes. PATIENTS AND METHODS: All children seen between 1982 and 1991 at the Pediatric Rheumatology Clinic at Newington Children's Hospital (Newington, CT) with an initial diagnosis of Lyme disease were identified. Medical records were reviewed and structured telephone interviews were conducted to obtain demographic, clinical, and follow-up data. RESULTS: A total of 90 children (63% boys) with a mean age of 8.3 years (range, 1.8-16 years) at the time of diagnosis of Lyme arthritis were evaluated. Lyme arthritis was preceded by early Lyme disease in 23 (26%) of the children; however, only 8 (35%) of these children had been treated with appropriate antimicrobial therapy at that early stage. Ninety percent of the children had arthritis of at least one knee, while small joints were rarely involved. For the 31 children who underwent arthrocentesis, the mean white blood cell count in the synovial fluid was 38 000 cells/mm3 (range, 7000-99 000 cells/mm3) with predominantly neutrophils. For the 79 children for whom an erythrocyte sedimentation rate was determined, the highest level for 61 (77%) was >20 mm/h and for 36 (46%) was >50 mm/h. Antimicrobial therapy was initiated 2 days to 5.5 years (median, 2 months) after the onset of symptoms. However, 5 of the children were never treated with antimicrobials. Fifty-one percent of the patients had a single episode of arthritis, while 49% reported recurrent episodes of arthritis over a period of 1 week to 8 years (median, 6 months). Two children (2%) developed chronic arthritis and underwent arthroscopic synovectomy. At the time of the telephone follow-up evaluation, performed 2 to 12 years (median, 7 years) after the onset of the Lyme arthritis, 4 children had ongoing musculoskeletal complaints that resulted in mild to moderate impairment of school or sports activities, but none of the children had evidence of active arthritis. CONCLUSION: The results of this investigation suggest that the prognosis for children with Lyme arthritis who are treated with appropriate antimicrobial therapy is excellent.

Early Lyme disease: a flu-like illness without erythema migrans.

The existence of a form of early Lyme disease characterized by a flu-like illness without erythema migrans is controversial. To confirm the existence and define the clinical characteristics of the flu-like illness without erythema migrans of localized Lyme disease, the authors studied patients from a Lyme disease endemic area of Connecticut who visited their primary care physicians with an undefined flu-like illness. Patients kept a diary of their symptoms. Acute and convalescent sera were obtained. The diagnosis of Lyme disease was based on the appearance of IgM or IgG antibodies to Borrelia burgdorferi as demonstrated by both enzyme-linked immunosorbent assay and immunoblot assay. Twenty-four untreated patients were studied. In five patients acute serologic evidence of Lyme disease developed. The flu-like illness in these five patients was characterized by fever and fatigue and resolved spontaneously in 5 to 21 days. Symptoms recurred in three of these five patients. The existence of a flu-like illness without erythema migrans of early Lyme disease has been clearly established. Prospective, controlled studies are needed to better define its incidence, characteristics, and prognosis so that appropriate diagnostic and therapeutic strategies can be developed.

Community-wide outbreak of group G streptococcal pharyngitis.

Although several outbreaks of group G beta-hemolytic streptococcal (GGBHS) pharyngitis have been described, doubt still remains regarding the etiologic role of GGBHS in acute pharyngitis beyond a limited number of situations. In the winter/spring of 1986/87, throat cultures were obtained from 222 consecutive children seen at a private pediatric office with acute pharyngitis and group A beta-hemolytic streptococci (GABHS) were recovered from 91 children (41%) and GGBHS from 56 children (25%). One patient had both GABHS and GGBHS isolated. This isolation rate of GGBHS was dramatically greater than in previous and subsequent years, and 67% of the GGBHS isolates occurred during an 8-week period. Results of DNA fingerprinting of the 57 isolates of GGBHS demonstrated that 43 (75%) appeared to be the same strain. The patients with GGBHS were comparable to those with GABHS with respect to clinical findings, antistreptolysin-O titer response, and clinical response to antibiotic therapy. However, patients with GGBHS were significantly older (P less than .05). This is the first well-documented, community-wide outbreak of GGBHS pharyngitis and the first respiratory outbreak of GGBHS pharyngitis in a pediatric population. GGBHS may be a more important cause of acute, treatable pharyngitis than had been previously recognized.

Potential mechanisms for failure to eradicate group A streptococci from the pharynx.

OBJECTIVE: To investigate the relative efficacy of orally administered cefadroxil and penicillin V in the treatment of group A streptococcal (GABHS) pharyngitis and the mechanism(s) responsible for failure of antimicrobial therapy to eradicate GABHS from the pharynx. STUDY DESIGN: A prospective, randomized clinical trial was conducted in four pediatric offices in which 462 patients with acute pharyngitis and positive culture for GABHS were randomly assigned to receive cefadroxil (n = 232) or penicillin V (n = 230). RESULTS: Bacteriologic treatment success rates for patients in cefadroxil and penicillin groups were 94% and 86%, respectively. However, among patients classified clinically as likely to have bona fide GABHS pharyngitis, there was no difference in bacteriologic treatment success rates in cefadroxil and penicillin groups (95% and 94%, respectively). Among patients classified clinically as likely to be streptococcal carriers, bacteriologic treatment success rates in cefadroxil and penicillin groups were 92% and 73%, respectively. The presence of beta-lactamase and/or bacteriocin-producing pharyngeal flora had no consistent effect on bacteriologic eradication rates among patients in either penicillin or cefadroxil treatment groups or among patients classified as having either GABHS pharyngitis or streptococcal carriage. CONCLUSIONS: Neither beta-lactamase nor bacteriocin produced by normal pharyngeal flora are related to bacteriologic treatment failures in GABHS pharyngitis. Cefadroxil seems to be more effective than penicillin V in eradicating GABHS from patients classified as more likely to be streptococcal carriers. However, among patients we classified as more likely to have bona fide GABHS pharyngitis, the effectiveness of cefadroxil and penicillin V seems to be comparable.

Histopathology of HCV infection.

The basic morphologic features of acute and chronic viral hepatitis C are similar to those of other hepatitides; however, hepatitis C is characterized by the histologic triad of lymphoid aggregates in portal tracts, epithelial damage of small bile ducts and microvesicular and macrovesicular steatosis of hepatocytes. Significant progress has been made in the demonstration of HCV in infected liver tissues by immunohistochemical and in situ hybridization techniques. The new classification of chronic hepatitis, based on etiology, grading (extent of necroinflammatory activity) and staging (extent of fibrosis) has been widely accepted and will lead to a better understanding of the variable course and response to therapy of this enigmatic disease.

A comparison of cefadroxil and penicillin V in the treatment of streptococcal pharyngitis in children.

The efficacy of cefadroxil, an orally administered broad spectrum cephalosporin, was compared with that of penicillin V in several studies comprising more than 550 children with group A beta-haemolytic streptococcal (GABHS) pharyngitis. Both drugs alleviated clinical signs and symptoms and eradicated GABHS from the upper respiratory tract within 18 to 24 hours of the initiation of therapy. Approximately 8% of the patients treated with either cefadroxil or penicillin V had strains of GABHS isolated from 1 of their follow-up throat cultures which were identical to the strains isolated from their initial throat cultures, and were considered bacteriological treatment failures. Compliance was greater than 90% with all of the regimens used, but was significantly better with cefadroxil given as a 30 mg/kg dose once daily than with penicillin V given 3 times daily. There were no serious adverse reactions with either drug. Thus, cefadroxil was shown to be well tolerated and as effective as the standard agent, oral penicillin V, in the treatment of GABHS pharyngitis in children.

Immunohistochemical findings in the renal vascular lesions of progressive systemic sclerosis.

Four selected patients with progressive systemic sclerosis showed deposition of immunoglobulins and complement in diseased renal arteries and arterioles. Although three patients had hypertension, in two of these malignant, the third patient did not have hypertension over a four year period. These findings suggest that immune complexes may be involved in the pathogenesis of some cases of progressive systemic sclerosis, the primary target being the vascular system.