End-of-life care following leg amputation in patients with peripheral artery disease or diabetes.

BACKGROUND: The aim was to characterize end-of-life care in patients who have had a leg amputated for peripheral artery disease (PAD) or diabetes. METHODS: This was a population-based retrospective cohort study of patients with PAD or diabetes who died in Ontario, Canada, between 2011 and 2017. Those who had a leg amputation within 3 years of death were compared with a control cohort of deceased patients with PAD or diabetes, but without leg amputation. The patients were identified from linked health records within the single-payer healthcare system. Place and cause of death, as well as health services and costs within 90 days of death, were compared between the amputee and control cohorts. Among amputees, multivariable regression models were used to characterize the association between receipt of home palliative care and in-hospital death, as well as time spent in hospital at the end of life. RESULTS: Compared with 213 300 controls, 3113 amputees were less likely to die at home (15·5 versus 24·9 per cent; P < 0·001) and spent a greater number of their last 90 days of life in hospital (median 19 versus 8 days; P < 0·001). Amputees also had higher end-of-life healthcare costs across all sectors. However, receipt of palliative care was less frequent among amputees than controls (inpatient: 13·4 versus 16·8 per cent, P < 0·001; home: 14·5 versus 23·8 per cent, P < 0·001). Among amputees, receipt of home palliative care was associated with a lower likelihood of in-hospital death (odds ratio 0·49, 95 per cent c.i. 0·40 to 0·60) and fewer days in hospital (rate ratio 0·84, 0·76 to 0·93). CONCLUSION: Palliative care is underused after amputation in patients with PAD or diabetes, and could contribute to reducing in-hospital death and time spent in hospital at the end of life.

ANTECEDENTES: Caracterizar la atención al final de la vida en pacientes con amputación de la extremidad inferior por enfermedad arterial periférica (peripheral arterial disease, PAD) o diabetes. MÉTODOS: Se trata de un estudio de cohortes retrospectivo de base poblacional en sujetos fallecidos con PAD o diabetes en Ontario, Canadá (2011-2017). A partir de los registros sanitarios incluidos en un sistema de salud de una sola entidad pagadora, se identificaron los individuos con amputación de la extremidad inferior en los 3 años previos al fallecimiento y una cohorte control de fallecidos con PAD o diabetes sin amputación. Entre las cohortes de amputados y controles se comparó el lugar del fallecimiento y la causa, así como el uso de servicios sanitarios y costes en los últimos 90 días de vida. En el grupo de los amputados, se utilizaron modelos de regresión para caracterizar la asociación entre recibir cuidados paliativos domiciliarios y el fallecimiento en el hospital, así como los días de estancia hospitalaria al final de la vida. RESULTADOS: En comparación con los controles (n = 213.300), los sujetos con amputación (n = 3.113) era menos probable que fallecieran en el domicilio (16% versus 25%, P < 0,001) y pasaron un mayor número de sus últimos 90 días de vida en el hospital (mediana 19 versus 8 días, P < 0,001). Los costes de atención sanitaria al final de la vida en todos los sectores también fueron mayores para los amputados. Sin embargo, recibir cuidados paliativos fue menos frecuente en los amputados que en los controles (en el hospital 13% versus 17%, P < 0,001; domiciliarios 14% versus 24%, P < 0,001). En el grupo de los amputados, recibir cuidados paliativos domiciliarios se asociaba con una menor probabilidad de fallecimiento en el hospital (razón de oportunidades, odds ratio 0,49, i.c. del 95% 0,40-0,60) y menos días de hospitalización (tasa de riesgo 0,84, i.c. del 95% 0,76-0,93). CONCLUSIÓN: Los cuidados paliativos están infrautilizados en pacientes con PAD o diabetes y pueden contribuir a disminuir los fallecimientos en el hospital y los días de hospitalización al final de la vida.

Pancreatic islet cell surface glycoproteins containing Gal beta 1-4GlcNAc-R identified by a cytotoxic monoclonal autoantibody.

To investigate the autoimmune pathogenesis of spontaneously occurring diabetes mellitus in BB rats, spleen cells of newly diagnosed diabetic BB rats were fused with mouse myeloma cells. Hybridoma supernatants were screened for antibodies by indirect immunofluorescence and by 51Cr-release assays using the RINm5F rat insulinoma cell line. One clone, E5C2, produced an IgM kappa antibody that was cytotoxic for RINm5F cells, but not for other rat cell lines nor for primary rat islet cells. However, treatment of primary rat islet cells with neuraminidase exposed surface antigens and rendered the cells susceptible to complement-mediated lysis by antibody E5C2. Using immunostaining of glycolipids separated by thin-layer chromatography, hapten inhibition assays with defined carbohydrates, and Western blots, the antigens recognized by E5C2 on RINm5F cells were identified as glycoproteins with molecular weights of 60,000 and 68,000. The antibody recognizes a carbohydrate antigen containing the sequence Gal beta 1-4GlcNAc-R, which on RINm5F cells is predominantly hidden by covalently bound sialic acid. These studies raise the possibility that hidden antigenic determinants on islet cells exposed by a variety of means may be the target of autoimmune attack.

Aspirin: an unexpected side effect on prostacyclin synthesis in cultured vascular smooth muscle cells.

Monolayer cultures of rat aorta smooth muscle cells synthesized the anti-aggregatory substance prostacyclin via the cyclooxygenase pathway from 14C-labeled arachidonic acid. The product was identified both by bioassay and by mass spectrometry. Labeled cells produced prostacyclin only when exposed to the initiator thrombin: treatment with therapeutic concentrations of aspirin (0.2 millimolar) for 30 minutes completely destroyed the cells' ability to synthesize prostacyclin. Prostacyclin synthesis from exogenous arachidonic acid recovered fully within 1 to 2 hours by a cycloheximide-sensitive process. Thrombin responsivness, which was permanently impaired in confluent nondividing cultures, recovered substantially and within 24 hours only when cells were stimulated to divide by subculturing. These results indicate that resting vascular cells can rapidly synthesize new cyclooxygenase, but that aspirin destroys additional components of the prostacyclin system which can only be replaced during cell division.

Anti-inflammatory drugs in experimental atherosclerosis. Part 4. Inhibition of atherosclerosis in vivo and thromboxane synthesis and platelet aggregation in vitro.

Groups of New Zealand white male rabbits were fed atherogenic diets containing 1% cholesterol. The diets of experimental groups were supplemented additionally with either aspirin, phenylbutazone, mefenamic acid, flufenamic acid, oxyphenylbutazone or aminopyrine. Blood cholesterol and phospholipids were measured at 3--4 week intervals. After 12 weeks the animals were sacrificed and the severity of atherosclerosis in the thoracic aorta was measured. In separate experiments, rabbit platelets were incubated with each of the drugs individually and conversion of [14C]arachidonic acid to thromboxanes and related compounds was assayed. Inhibition of collagen and arachidonic acid-induced platelet aggregation by each drug was also measured. All drugs inhibited thromboxane synthesis and platelet aggregation in varying degrees with flufenamate and aspirin being most and aminopyrine least effective. The pattern of metabolite formation from [14C]arachidonate was consistent with a block in the cyclooxygenase reaction. Phenylbutazone, flufenamic acid and oxyphenylbutazone produced significant reductions in atherosclerotic plaque formation without major changes in blood cholesterol levels or blood cholesterol--phospholipid ratios. Aspirin and aminopyrine were ineffective. The results indicate that the effectiveness of anti-inflammatory drugs as inhibitors of thromboxane synthesis and platelet aggregation in vitro does not afford a sufficient predictive index of their anti-atherogenicity in vivo. The significance of these findings is discussed in terms of the possible involvement of cyclooxygenase derivatives in atherogenesis.

5-Aminosalicylate: oxidation by activated leukocytes and protection of cultured cells from oxidative damage.

It has been postulated that oxygen radicals may play a role in the pathogenesis of inflammatory bowel disease. If so, then a drug like 5-aminosalicylate (5-ASA), which is used to treat such diseases, might work by interacting with oxygen-derived species. We found that activated mononuclear cells and activated granulocytes, as well as the products of the Fenton reaction, transformed [14C]5-ASA to a number of metabolites, among which we have characterized salicylate and gentisate. We also found that the lethal effect on cultured Chinese hamster ovary cells of adding either superoxide radical or hydrogen peroxide, components of the respiratory burst of activated white blood cells, was diminished by the addition of 100 micrograms/ml (0.65 mM) of 5-ASA. Thus, we have demonstrated that 5-ASA was oxidized by the oxidative burst of white blood cells and that 5-ASA protected cells from damage by oxygen-derived species, two findings which may offer an explanation for the role of 5-ASA in the treatment of inflammatory bowel disease.

Stable expression of mouse Cyp1a1 and human CYP1A2 cDNAs transfected into mouse hepatoma cells lacking detectable P450 enzyme activity.

Using the mouse hepatoma Hepa-1c1c7 c37 mutant cell line that exhibits negligible benzo[a]pyrene hydroxylase (Cyp1a1) and acetanilide 4-hydroxylase (Cyp1a2) enzyme activities, we developed stable transfectants of plasmids containing the murine Cyp1a1 (cytochrome P(1)450) and the human CYP1A2 (P(3)450) cDNAs. We show that the assay measuring metabolism of ethoxyfluorescein ethyl ester (EFEE) was invaluable in screening large numbers of individual cell lines for high Cyp1a1 enzyme activity. Nine different plasmid constructs containing various combinations of promoter and enhancer sequences were compared, including: the Drosophila heat shock promoter, the mouse mammary tumor virus long terminal repeat (MMTV LTR) carrying the glucocorticoid-responsive element (GRE), enhancer sequences from simian virus 40 (SV40) and herpes simplex virus type 1 (HSV-1), and the aromatic hydrocarbon-responsive domain (AhRD) of the murine Cyp1a1 gene. Interestingly, only those constructs containing the AhRD produced high levels of Cyp1a1 enzyme activity. In contrast, high levels of CYP1A2 activity were obtained with plasmids carrying the HSV-1 enhancer, as well as the AhRD. These studies suggest that the AhRD, which responds to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), provides a post-transcriptional signal necessary for the induction of functional Cyp1a1 enzyme activity. Although untransfected c37 cells exhibit markedly elevated levels of endogenous Cyp1a1 mRNA, the expression of exogenous Cyp1a1 or CYP1A2 enzyme activity in these cells decreases the concentration of this endogenous Cyp1a1 mRNA to negligible levels and restores Cyp1a1 mRNA inducibility by TCDD; these data indicate that the functional product of either the Cyp1a1 gene or the CYP1A2 gene might have a role in an autoregulatory loop controlling the constitutive expression of the Cyp1a1 gene. The cell lines described herein should be valuable in assessing the contribution of these two P450 enzymes to the processes of cytotoxicity, mutagenesis, and carcinogenesis.

Rickettsia tsutsugamushi infection in cell culture: antibiotic susceptibility determined by flow cytometry.

Recent unpublished reports from northern Thailand of severe and sometimes fatal cases of scrub typhus, despite appropriate antibiotic therapy, suggest that resistance may occur. Current antibiotic susceptibility methods that use direct microscopic counts of Giemsa-stained cells or mouse protection assays are slow, labor-intensive, and expensive. We explored the use of flow cytometry to measure rickettsial infection in vitro in L-929 cells treated with and without doxycycline, ciprofloxacin, erythromycin, and chloramphenicol. It was possible to detect the rickettsiae down to a level of 83% of the cells infected, mean of 37 rickettsiae per cell, and 40% of cells with too many rickettsiae to count. This level of sensitivity was sufficient to determine the inhibitory effect of all four drugs at standard screening concentrations. At lower concentrations of doxycycline, flow cytometry detected inhibition of rickettsial growth at a concentration of 6.25 x 10(-2) micrograms/ml but not at 6.25 x 10(-3) micrograms/ml, suggesting that the minimum inhibitory concentration is somewhere between these two values. The data from this study show that flow cytometry permits the rapid screening of numerous rickettsial isolates for their susceptibility to a variety of antibiotics, but that visual counts of infected cells provide a more precise indication of rickettsial growth.

Leukocyte adhesion deficiency: report of a case and review of the literature.

Leukocyte adhesion deficiency (LAD) is a rare inherited immunodeficiency that is characterized by deficiency of the beta 2 integrin leukocyte adhesion molecules Mac-1, LFA-1, and p150,95. We describe a case of the severe form of LAD in an infant with recurrent infections and with a complete deficiency of beta 2 integrin molecules, and review the clinical aspects of the syndrome.

Ceramide increases steroid hormone production in MA-10 Leydig cells.

Ceramide is known to have major roles in the control of cell proliferation, differentiation, and apoptosis. Recent studies also have shown that ceramide affects steroid production by JEG-3 choriocarcinoma cells, acutely dispersed rat Leydig cells, and ovarian granulosa cells, but the mechanism by which this occurs is unknown. Because ceramide induces apoptosis in many different cell types, we hypothesized that ceramide might affect steroidogenesis and/or induce apoptosis in MA-10 murine Leydig cells. To test this, MA-10 cells were incubated with either the water soluble C2-ceramide, (N-acetyl-sphingosine, 0.01-10 cm); bacterial sphingomyelinase (1-100 mU/ml); or C2-dihydroceramide (N-acetyl-sphinganine, 0.1-10 microM). The data show that N-acetyl-sphingosine significantly increased basal (0.87 +/- 0.2 vs. 0.42 +/- 0.09 ng/mg cell protein, P < 0.01) and human chorionic gonadotropin (hCG) stimulated progesterone (P) synthesis (204 +/- 12 vs. 120 +/- 5 ng/mg cell protein, P < 0.001); as did sphingomyelinase (basal P = 0.83 +/- 0.1 ng/mg cell protein, P < 0.01; hCG stimulated P = 173 +/- 7 ng/mg cell protein, P < 0.001). C2-dihydroceramide also increased basal P synthesis but was less effective than ceramide on a molar basis. Neither sphingomyelinase (100 mU/ml) nor ceramide (10 microM) had any effect on cAMP production or human chorionic gonadotropin binding; and neither induced any signs of apoptosis (FragEL DNA fragmentation assay and electron microscopy). Cells incubated with anti-Fas (300 ng/ml) demonstrated DNA fragmentation, nuclear condensation, and frequent apoptotic bodies, but had no change in P synthesis. These data show that ceramide significantly increases MA-10 Leydig cell P synthesis but does not induce apoptosis. The mechanism by which ceramide increases steroid hormone synthesis remains unknown but does not appear to be linked to the induction of apoptosis in MA-10 cells.

Sphingomyelinase inhibits in vitro Leydig cell function.

Activation of the immune system has profound effects on endocrine function which are mediated by cytokines including tumor necrosis factor-alpha (TNF alpha). In vitro, TNF alpha has been shown to directly inhibit Leydig cell testosterone (T) production, but the mechanism of this effect is still unclear. Recent studies using cultured human fibroblasts have shown that TNF alpha stimulates the activity of neutral sphingomyelinase (SMase) which hydrolyses sphingomyelin (SM) generating ceramide and changing membrane components including cholesterol. The cellular affects of increased SMase activity have been reproduced in vitro by the addition of exogenous SMase. In cultured fibroblasts, exogenous SMase decreases cholesterol synthesis. These findings led us to hypothesize that SMase might be important in the regulation of steroid hormone synthesis. To our knowledge, no previous studies have investigated this possibility. To test this hypothesis, rat Leydig cell enriched cultures were incubated in media containing SMase (0.1 to 100 mU/ml) or in control media. SMase significantly decreased basal and human chorionic gonadotropin (hCG) stimulated T production. SMase also decreased hCG binding and hCG stimulated adenosine 3':5'-cyclic monophosphate (cAMP). N-acetyl-sphingosine (0.1 to 10 microM), a water soluble ceramide, was used to determine whether or not the effects of SMase could be reproduced by ceramide addition. N-acetyl-sphingosine had only slight effects on basal T and cAMP, and no effect on hCG binding or hCG stimulated T or cAMP. These data suggest the metabolism of membrane sphingomyelin may be an important regulatory pathway in the control of Leydig cell function.

Restoration of prostacyclin synthase in vascular smooth muscle cells after aspirin treatment: regulation by epidermal growth factor.

Prostacyclin is a potent vasodilator and inhibitor of platelet aggregation and plays an important role in maintenance of vascular homeostasis. Aspirin irreversibly inactivates prostacyclin synthetase by acetylating the enzyme. Recovery of the enzyme following inactivation by aspirin was studied in rat aorta smooth muscle cells in tissue culture. Confluent cultures superfused with [14C]arachidonic acid, synthesized prostacyclin (PGI2) together with prostaglandins E2, D2, and F2 alpha. Brief treatment with physiological levels of aspirin (0.2 mM) completely inactivated prostacyclin synthesis. Following aspirin removal and addition of fresh growth medium, PGI2 synthesis recovered rapidly with a T 1/2 of only 30-40 min, compared to a doubling time of 24-30 hr for the cells. Recovery of PGE2, PGD2, and PGF2a synthesis paralleled that of PGI2, confirming that cyclooxygenase rather than endoperoxide-prostacyclin isomerase was the labile component. Recovery of PGE2 synthesis after aspirin was blocked by cycloheximide but not by actinomycin D. Recovery of aspirin-inactivated cells required a non-dialyzable component present in serum. All samples tested, including fetal bovine, new-born calf, human, and guinea pig, showed the activity. Fresh serum also induced a cycloheximide-sensitive 2- to 3-fold increase in cyclooxygenase levels in resting confluent cells within 1 to 2 hr. Serum factor was also required to restore PG synthesis after aspirin-inactivation in other cells, including 3T3 mouse fibroblasts, SV40-3T3 and K-Balb 3T3 transformed mouse fibroblasts, NRK rat kidney cells, and REF-9 rat embryonic fibroblasts. The activity was thermolabile, and was completely removed from the medium by growing cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of enzyme levels in the blood. Influence of environmental and genetic factors on enzyme clearance.

Since its discovery, lactic dehydrogenase virus (LDV) has remained unique as a model of long-term enzyme elevation due to impairment of enzyme clearance. The present study shows that mice inoculated with silica develop an increase in plasma lactate dehydrogenase (LDH) lasting for at least 6 months and that the enzyme elevation is due, at least in part, to impairment of clearance. The extent of the enzyme elevation is dependent on both the dose and route of silica administration and mice that had received both silica and LDV showed a more profound impairment of LDH clearance than mice that had received silica or LDV alone. Examination of the factors that regulate circulating enzyme levels in normal mice revealed that whereas there was no difference in resting enzyme levels among several inbred strains of mice (BALB/cAnN, NZBWF1/J,B10.D2/nSnN, and A/J mice), when mice were stressed by the administration of an enzyme load, certain inbred strains (BALB/cAnN) cleared the enzyme rapidly and others (B10.D2/nSnN) cleared the enzyme slowly. Moreover, in B10.D2/nSnN mice, enzyme clearance was age-related. When different strains of mice were infected with LDV, LDH levels were substantially higher in the circulation of slow enzyme clearers as compared to rapid enzyme clearers. It is concluded that both environmental and genetic factors influence the clearance of LDH and that impairment of enzyme clearance may be a more important factor than previously suspected in regulating enzyme levels in disease states.

In vitro effectiveness of azithromycin against doxycycline-resistant and -susceptible strains of Rickettsia tsutsugamushi, etiologic agent of scrub typhus.

In an effort to find a potential alternative treatment for scrub typhus, we evaluated the effectiveness of the standard drug doxycycline and the new macrolide azithromycin against a doxycycline-susceptible strain (Karp) and a doxycycline-resistant strain (AFSC-4) of Rickettsia tsutsugamushi. The antibiotics were tested in an in vitro assay system in which infected mouse fibroblast cells (L929) were incubated for 3 days in various concentrations of the drugs. Rickettsial growth was evaluated by direct visual counts of rickettsiae in Giemsastained cells or by flow cytometry. Initial tests were conducted at the concentration of each antibiotic considered to be the upper breakpoint for susceptibility (16 micrograms/ml for doxycycline and 8 micrograms/ml for azithromycin). Growth of both Karp and AFSC-4 was strongly inhibited with both antibiotics, as measured by visual counts, although the percentage of cells infected with AFSC-4 in the presence of doxycycline was three times greater than the percentage of cells infected with Karp but was only 60% as great as the percentage of cells infected with Karp in the presence of azithromycin. Flow cytometry confirmed that rickettsial growth occurred in the absence of antibiotics, but it failed to detect it in the presence of high concentrations of either drug. Visual counts of rickettsial growth at lower concentrations of the antibiotics (0.25 to 0.0078 microgram/ml) showed that the Karp strain was 16 times more susceptible that the AFSC-4 strain to doxycycline. Azithromycin was much more effective than doxycycline against AFSC-4, inhibiting rickettsial growth at 0.0156 microgram/ml to levels below that achieved by 0.25 microgram of doxycycline per ml. Azithromycin was also more effective than doxycycline against the Karp strain, causing greater reductions in the number of rickettsiae per cell at lower concentrations. If in vivo testing confirms the in vitro effectiveness of azithromycin, it may prove to be the drug of choice for the treatment of scrub typhus in children and pregnant women, who should not take doxycycline, and in patients with refractory disease from locations where doxycycline-resistant strains of R. tsutsugamushi have been found. When tested in an in vitro assay system, azithromycin was more effective than doxycycline against doxycycline-susceptible and -resistant strains of R. tsutsugamushi.

Tannic acid's effect on bird antigen.

BACKGROUND: Birds have been associated with many diseases including hypersensitivity pneumonitis and allergic diseases such as asthma and rhinitis. Bird antigen from homes of patients with hypersensitivity pneumonitis persists long after the bird is removed from the home. This may account for the persistence of symptoms, signs, and bird-specific IgG in patients with hypersensitivity pneumonitis. Tannic acid application has been effective in decreasing cat and mite allergen levels. No data have been available on tannic acid's effect on bird antigen. OBJECTIVE: It is the purpose of this study to determine whether tannic acid reduces bird antigen in the home. METHOD: Dust samples were collected from homes with bird antigen before and after application of tannic acid. Samples were assayed for bird antigen levels using a competitive inhibition ELISA. Pre- and post-bird antigen levels were compared using a paired t test to determine whether antigen was reduced significantly. RESULTS: There was not a statistical difference between bird antigen levels before and after application of tannic acid as compared by paired t test (P = .09). CONCLUSION: Tannic acid is not effective in decreasing bird antigen levels. In patients with hypersensitivity pneumonitis or allergic disease to birds, the bird should be removed from the home and environmental cleanup should be undertaken, but tannic acid application is not indicated.

Flow cytometry to identify cell types to which enzymes bind. Effect of lactic dehydrogenase virus on enzyme binding.

Flow cytometry was used to measure the binding of enzymes (i.e. lactate dehydrogenases 1 and 5, malate dehydrogenase, and asparaginase) to cells. Of the four enzymes studied, asparaginase showed the greatest binding. Single color analysis revealed that asparaginase bound best to preparations enriched in macrophages, and dual color analysis showed that the binding was to macrophages. Studies on continuous cell lines revealed that asparaginase bound to one mouse macrophage line, but not to another or to murine fibroblasts. Inoculation of mice with lactic dehydrogenase virus, a virus that infects macrophages, decreased the in vivo clearance of asparaginase from the circulation and the in vitro binding of asparaginase to peritoneal macrophages. It is concluded that flow cytometry can be used to study the binding of enzymes to cells, to identify the cell type to which the enzyme binds, and to measure changes in the capacity of cells to bind enzymes.

Recovery and viability of Orientia tsutsugamushi from packed red cells and the danger of acquiring scrub typhus from blood transfusion.

BACKGROUND: The purpose of this study was to determine whether infective Orientia tsutsugamushi, the etiologic agent of scrub typhus, could survive normal blood banking processing and storage procedures. STUDY DESIGN AND METHODS: Mononuclear cells isolated from whole blood by density gradient centrifugation were inoculated with O. tsutsugamushi, Karp strain. Infection of the mononuclear cells was confirmed by Giemsa stain, direct fluorescent antibody assay, and polymerase chain reaction using primers specific for the groESL operon of O. tsutsugamushi. The quantity of rickettsial particles in each preparation was determined by direct counts from the Giemsa-stained preparations. Infected mononuclear cells were returned to their respective aliquots of packed red blood cells, which were then either stored at 4 degrees C or glycerolized and frozen at -70 degrees C. RESULTS: Rickettsiae survived up to 10 days (but not 30 days) of refrigerated storage and 45 days of frozen storage, as determined by inoculation of mice with 0.5-mL aliquots of the blood components. Infection of the mice was determined by illness, death, direct fluorescent antibody assay of peritoneal smears, polymerase chain reaction of blood, and enzyme-linked immunosorbent assay detection of antibodies in plasma. CONCLUSION: Because the quantity of rickettsiae injected into the mice was comparable to the quantity reported in the literature for human blood during natural infections, scrub typhus could present a risk in blood collected from donors in endemic areas. This may especially be true, because people can be rickettsemic before illness, after successful antibiotic treatment, and chronically after resolution of disease.

Human monoclonal multiple-organ-reactive autoantibodies distinguished by mouse monoclonal anti-idiotypic antibodies: expression of idiotopes in humans with and without autoimmune diseases.

Previously we reported on the production and characteristics of a number of human monoclonal autoantibodies. All of these autoantibodies were of the IgM class and reacted with antigens in multiple organs. In this study we generated IgG murine monoclonal anti-idiotypic antibodies against five human monoclonal autoantibodies, (i.e., MOR-h2, MOR-h3, MOR-h4, CG1, and CG2). These anti-idiotypic antibodies reacted strongly with the corresponding human monoclonal autoantibody, but minimally or not at all with other human monoclonal autoantibodies. By using these anti-idiotypic antibodies as probes, we screened sera obtained from normal individuals and patients with insulin-dependent diabetes mellitus, Hashimoto's thyroiditis, and systemic lupus erythematosus for the expression of idiotopes. Our study showed that the idiotopes recognized by three of the anti-idiotypic antibodies, i.e., anti-CG1, anti-CG2, and anti-MOR-h2, were not expressed, but the idiotopes recognized by two of the anti-idiotypic antibodies, i.e., anti-MOR-h3 and anti-MOR-h4, were expressed in normal individuals. In patients with autoimmune disorders, there was no increase in the expression of the CG1, CG2, and MOR-h2 idiotopes, but 45 and 23% of the patients with systemic lupus erythematosus showed a significant increase in the expression of the MOR-h3 and MOR-h4 idiotopes respectively. These findings show that there is widespread expression in the B cell repertoire of certain autoantibody-associated idiotopes.

Serum autoantibodies in type 1 (insulin-dependent) diabetes mellitus: identification of reactivity against a 29 kd pancreas-specific protein.

Serum samples from patients with type 1 (insulin-dependent) diabetes mellitus and controls were incubated with two-dimensional Western immunoblots of pancreas and other tissues. Two out of 26 (8%) of the diabetics and 0 out of 45 of the controls demonstrated reactivity against four pancreas-specific proteins with identical molecular weights of 29,000 daltons and different isoelectric points ranging from pH 7.0-8.0. It is concluded that 29 Kd autoantibody reactivity is not a major marker for type 1 diabetes, but may help identify a subgroup of type 1 diabetics.