Acid pH-induced changes in the immunoreactivity of specific antigen and antibody in circulating immune complexes from tuberculosis sera.

The effect of acid pH treatment of circulating immune complexes (CIC) derived from tuberculosis sera was studied on the relative titers of specific antibody (CIC Ab) and mycobacterial antigen (CIC Ag) in the complexes. While the specific antibody titers increased, the titers of CIC Ag declined as a result of acid pH treatment of CIC, both changes being highly significant statistically. Direct exposure of TB sera to pH 2.8 also resulted in significant enhancements in the titers of antibodies directed against Mycobacterium tuberculosis (M.tb.). Competition experiments indicated that the acid pH-treated antibodies retained their antigen specificity. TB sera treated with pH 2.8 for 30 min and then neutralized back to pH 7.4 retained the enhanced antibody reactivity even after 7 days of storage at 4 degrees C. Our results indicate that acid pH treatment induces higher antigen binding ability in anti-M.tb. antibodies, present in free or complexed form in TB sera. These changes appear to be irreversible. Dissociation of CIC and analyzing the titers of released antibody and antigen components offered no advantage with respect to the immunodiagnosis of tuberculosis, as compared to the levels of undissociated CIC components or the serum antibody.


Novel and ultrasensitive noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay) for alpha-human atrial natriuretic peptide.

A novel and ultrasensitive noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay) for alpha-human atrial natriuretic peptide (alpha-hANP) in plasma is described. alpha-hANP was biotinylated using N-hydroxysuccinimidobiotin and trapped onto an anti-alpha-hANP [6-28] IgG-coated polystyrene ball. After washing, biotinylated alpha-hANP was eluted from the polystyrene ball with HCI and was reacted with 2,4-dinitrophenyl-fluorescein-bovine serum albumin-disulfide-rabbit anti-alpha-hANP [6-28] IgG conjugate. The complex formed was trapped onto (anti-2,4-dinitrophenyl group) IgG-coated polystyrene balls and, after washing, reacted with avidin-beta-D-galactosidase conjugate. The polystyrene balls were washed, and the complex of the three components was eluted with epsilon N-2, 4-dinitrophenyl-L-lysine and transferred to anti-fluorescein IgG-coated polystyrene balls. After washing, the complex was released from the polystyrene balls by reduction with 2-mercaptoethylamine and transferred to (anti-rabbit IgG) IgG-coated polystyrene balls. beta-D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. The detection limit of alpha-hANP [1-28] was 3 fg (1 amol)/tube. Interference by plasma proteins was eliminated by separation of peptides from proteins using a molecular sieve. The assay range of plasma alpha-hANP [1-28] was 0.04-120 ng/L, and plasma levels of hANP in healthy subjects (11-56 ng/L) were measured without concentration.


Comparison of serum amyloid A and C-reactive protein as indicators of lung inflammation in corticosteroid treated and non-corticosteroid treated cystic fibrosis patients.

Serum amyloid A (SAA) and C-reactive protein (CRP) levels were compared in 830 serum samples from 155 cystic fibrosis (CF) patients. Correlation coefficients were calculated for all samples (r = 0.73), for samples from non-corticosteroid treated (CFNS) patients (n = 698, r = 0.80), and for samples from corticosteroid treated (CFS) patients (n = 132, r = 0.35). SAA was the more sensitive indicator of pulmonary inflammation when SAA and CRP were compared to pulmonary function tests of 49 hospitalized patients at admission and discharge. CRP levels were significantly (p less than .05) lower at admission in CFS patients than in CFNS patients, whereas SAA levels were not significantly different between the two groups. All nine CFS patients hospitalized had elevated SAA levels (average 22 times above normal limits) at admission, while only six had elevated CRP levels (average 3.7 times above normal limits) at admission. In the 40 CFNS patients both SAA and CRP levels were significantly elevated at admission. In each case SAA and CRP levels declined as pulmonary functions improved with effective antimicrobial therapy. In three instances SAA levels increased during hospitalization while CRP levels did not. In each case, rising SAA levels indicated clinical deterioration associated with evolving resistance of P. aeruginosa which required a change in antibiotic therapy.


A new sensitive microplate assay of plasma endotoxin.

We have developed a microplate method for determining endotoxin in platelet-rich plasma-using Endospecy, an endotoxin-specific chromogenic Limulus test reagent. Nonspecific activators and inhibitors of the test were eliminated by exposing samples (5 microliters) to the alkali reagent consisting of KOH, CaCl2, Triton X-100, ethyleniminepolymer and N,N-bis(2-hydroxyethyl)glycine. The recoveries of various endotoxins were almost complete and not enhanced by dilution. The dose-response curve was linear over endotoxin concentrations of 2-400 pg/ml with good precision (C.V. less than 5.0%). Normal human plasmas (n = 30) contained less than 5.0 pg/ml of endotoxin in reference to that of Escherichia coli 0111: B4. All plasma samples with high concentration of endotoxin by a conventional method showed high values by the microplate assay as well. Since it does not require centrifugation, the new treatment allows the whole reactions to proceed on the same microplate. This permits us to apply the Limulus test to an automated assay system, making plasma endotoxin determination simpler and more rapid than a conventional test tube method.


Automated quantitative nephelometric latex immunoassay for determining ferritin in human serum.

We describe a rapid and sensitive latex nephelometric immunoassay for quantifying ferritin in human serum. This latex immunoassay procedure uses commercially available ready-for-use reagents [Tina-Quant (a) Ferritin, Boehringer Mannheim] that have a long shelf life. The assay consists of incubating the diluted serum sample (5-fold) for 12 min at room temperature with latex particles covalently coated with anti-ferritin antibodies, and then quantifying the change of light-scatter produced. The assay is fully automated on the Behring nephelometer analyzer with a sampling rate of 150 samples/hour. The method has an analytical range of 3 to 260 micrograms/l. Maximal intra- and inter-assay CVs were 4.0 and 6.2%, respectively. Analytical recoveries ranged from 91.3 to 103.6%. Assay detection limit was less than 3 micrograms/l. Linearity of the test is given throughout the measuring range. There was no interference from bilirubin (up to 340 mumol/l), haemoglobin (up to 7 g/l), or rheumatoid factor (up to 1,100 IU/ml). Turbid and lipemic samples interfere. This interference may be avoided by pretreating these samples prior to assay. Results correlated well with those obtained by an automated ELISA test (r = 0.995) and with those of two commercial RIA methods (r greater than 0.97). This latex nephelometric procedure is a convenient method and represents an interesting alternative to other immunoassays for measuring ferritin in human serum.


User-defined serum aspartate and alanine aminotransferase, cholesterol, triglycerides, urea, and uric acid for the Beckman synchron CX 4/5 using Ames Sera-Pak reagents.

Beckman aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholesterol, triglycerides, urea, and uric acid Liquid Reagents for Synchron CX 4/5 (48, 48, 25, 60, 26, and 30 cents US/test, respectively) are expensive. We have established our own methods for serum AST, ALT, cholesterol, triglycerides, urea, and uric acid (6, 6, 5, 12, 13, and 6 cents US/test, respectively) using Ames Sera-Pak reagents. Linearity of our AST, ALT, cholesterol, triglycerides, urea, and uric acid methods were either similar to or higher than the Beckman methods. The within run and day-to-day run precisions were acceptable. Recovery of our AST, ALT, cholesterol, triglycerides, urea, and uric acid were excellent. Our results for AST, ALT, cholesterol, triglycerides, urea, and uric acid correlated well with the Beckman results. Bilirubin (340.8 mumol/L) did not significantly interfere on our AST, ALT, cholesterol, triglycerides, and urea, while its concentrations of 165.8 mumol/L started giving negative interference on uric acid. Turbidity (2+) did not interfere significantly on our AST and ALT but started giving positive interference on cholesterol, triglycerides, urea, and uric acid. Hemolysis (2+) gave positive interference on our cholesterol, triglycerides, urea, and uric acid. Stability of Ames Sera-Pak working reagents was at least 30 days for AST, ALT, urea, and uric acid and 40 days for cholesterol and triglycerides.


Evaluation of the clinical utility of platelet aggregation studies in the long-term follow-up of patients with atherosclerotic vascular disease.

The present study was designed to evaluate the usefulness of laboratory monitoring of antiplatelet therapy by means of a multiparametric evaluation of in vitro platelet aggregation tests in the attempt to individually optimize a given therapeutic regimen. The presence of a condition of hyperaggregability was shown in approximately 80% of patients with different forms of atherosclerotic vascular disease not undergoing any therapeutic regimen with antiplatelet agents. Conversely, a significant decrease in platelet activity was observed in patients undergoing different therapies based on acetylsalicylic acid (ASA), ticlopidine, or indobufen. The similar antiaggregatory effect of low-dose vs. high-dose ASA therapies was also shown. Dipyridamole alone showed no antiaggregatory effect, which, in turn, was reached only by the addition of ASA. Nevertheless, the association of ASA plus dipyridamole did not show any stronger antiplatelet effect than ASA alone. The evaluation of in vitro platelet activity in a group of patients treated with picotamide failed to show any significant change in comparison with the untreated group, probably due to the short half-life of picotamide in man and/or to its capability of reversibly antagonizing the action of thromboxane at receptor level. The evaluation of a long-term follow-up of 90 patients treated with different antiplatelet agents supports the idea that a multiparametric analysis of in vitro platelet aggregation may provide valuable help in monitoring and optimizing a given therapeutic regimen.


Determination of aluminum in biological fluids by furnace atomic absorption spectrophotometry.

Detailed procedures were developed for the furnace atomic absorption spectrophotometry (FAAS) determination of aluminum (Al) in serum, urine, cerebrospinal fluid (CSF), and proportionated dialysate. Of particular note were the use of Mg (NO3)2.6H2O as a matrix modifier and the employment of the standard additions routine in analysis. The accuracy of the method(s) used is supported by work with assayed controls and by recovery studies. The use of a "clean room" was shown to be unnecessary. Normal serum, urine, and CSF Al ranges observed were 4.8-8.9, 5.1-9.1, and 1.0-5.8 micrograms L-1 respectively.


Contemporary concepts of autoimmunity and autoimmune diseases.

No single theory or mechanism can explain the phenomenon of autoimmunity and autoimmune diseases. Not all autoimmune responses are harmful or "forbidden." Considerable research has indicated that autoimmune response may be normal and important in the regulation of the immune system. Autoimmunity may play a role in a wide range of clinical states including physiological clearance of dead cells, and cell components, aging, response to viral and microbial infections, and generalized immunological diseases. There are many factors involved in autoimmunity including genetic, hormonal, immunological, and environmental factors. The susceptibility to autoimmune diseases is multifactorial and polygenic. There is a definite association of the autoimmune diseases with MHC alleles. Also, non-MHC genes are involved in disease susceptibility. Numerous mechanisms of autoimmunity have been discussed. There may be an alteration with dysregulation of the immune system with defective generation of normal suppressor mechanisms or an altered neuroendocrine regulation. The altered immune system will make the host more susceptible to autoimmune disease. Autoimmune reactions can occur in a host with a normal immune system. Some examples are as follows: 1. Infection or damage to host target organ with release or alteration of autoantigen 2. Molecular mimicry or cross-reactivity between virus or bacteria and host autoantigens 3. Abnormal expression of MHC molecules by antigen-presenting cells in target cells resulting in activation of autoreactive T-cells. 4. Drug administration


Rapid screening method for polymorphism of group A apolipoproteins.

Polymorphism of apolipoproteins AI and AII (apo AI and apo AII) can be easily investigated in plasma by a simple method involving a 30-min incubation of EDTA plasma in the presence of urea, dithiothreitol, and Nonidet P-40 followed by subsequent isoelectric focusing (IEF). The sample (2 microL) was applied to an ultrathin flat acrylamide gel of pH range 4-6, and focused using a Bio-Rad Mini IEF Cell for 1.5 h at a maximum of 500 V. Coomassie Blue R-250 was used to visualize the apolipoproteins. To verify the identity of the different apolipoproteins after IEF, the gel was immunofixed directly with anti-apo AI, or immunoblotted on polyvinylidene difluoride (PVDF) membrane using monospecific antibodies to apo AI and apo AII and an anti-immunoglobulin-alkaline phosphatase conjugate. High-density lipoprotein (HDL) was used as a standard for Apo AI variants. Employing these techniques, human plasma apo AI was resolved into one major band (apo AI0, pI 5.54), and four minor bands identified as apo AI+2 (pI 5.75), apo AI+1 (pI 5.66), apo AI-1 (pI 5.45), and apo AI-2 (pI 5.34). Apo AII was resolved into one major isoprotein designated as apo AII0 (pI 4.87), and two minor isoforms apo AII+1 and apo AII-1 which focused at pIs of 5.18 and 4.58, respectively. The results showed that these methods can be used to identify apo AI and AII isoforms without prior ultracentrifugation to isolate the HDL. The entire procedure, including IEF, fixation (chemical or immunofixation), and staining, can be accomplished in 5 h compared to 2 days using previously reported technique.(ABSTRACT TRUNCATED AT 250 WORDS)


Sera from simian immunodeficiency virus-infected rhesus macaques inhibit lymphocyte proliferation.

Rhesus macaque monkeys infected with the simian immunodeficiency virus (SIV) develop a syndrome mimicking acquired immunodeficiency syndrome (AIDS) in humans. We had demonstrated previously that sera from individuals infected with human immunodeficiency virus (HIV) inhibit the proliferation of lymphocytes from healthy noninfected subjects and that this phenomenon was associated with the development of clinical AIDS. Thus, we sought to determine whether sera from SIV-infected monkeys would also inhibit lymphocytes from healthy humans and SIV-negative rhesus monkeys. Sera from SIV-infected monkeys were compared with sera from uninfected animals and cultured with cells from healthy human volunteers or SIV-negative monkeys in the presence or absence of phytohemagglutinin (PHA). Cell proliferation was determined by measuring the incorporation of radiolabeled thymidine into cellular DNA. Sera from SIV-infected monkeys suppressed the proliferation of human and non-human primate lymphocytes. This activity appears to be similar to that described for sera from HIV-1-infected humans. Therefore, rhesus macaques infected with SIV provide a model for the study of serum inhibitory factors previously reported in AIDS patients.


Sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG in serum using recombinant gag p24(14-214) as antigen.

A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using recombinant gag p24(14-214) of HTLV-I is described. The recombinant gag p24(14-214) is soluble in the absence of detergents and allows the use of enzymes other than horseradish peroxidase as a label in the assays. The usefulness of recombinant gag p24(14-214) was examined with 305 sera characterized by other methods including gelatin particle agglutination, enzyme-linked immunosorbent assay (ELISA) using HTLV-I, and Western blotting. This assay was more sensitive than other methods using HTLV-I as antigen. The specificity could be tested by preincubation of test serum with excess of the recombinant protein. Most of negative and positive sera were discriminated. However, some results appeared to be false-positive or false-negative, and recombinant gag p24(14-214) was suggested to be useful, when used with other recombinant proteins and/or peptides, for improving the reliability of serodiagnosis by separately demonstrating antibodies against as many different epitopes of HTLV-I as possible. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant gag p24(14-214) conjugate and recombinant gag p24(14-214)-beta-D-galactosidase conjugate. The complex formed consisting of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG.(ABSTRACT TRUNCATED AT 250 WORDS)


Impaired cytokine production in whole blood cell cultures from patients with colorectal carcinomas as compared to benign colorectal tumors and controls.

Cytokine production was investigated in whole blood cell cultures from 74 patients with colorectal carcinomas, 20 patients with benign colorectal tumors, and 314 healthy controls. In the 4 day post induction supernatants the levels of IFN-gamma, IL-1-alpha, IL-2, and TNF-alpha were measured by a sensitive immunoassay. In the blood cell cultures of the patients with colorectal carcinomas significantly lower values of IFN-gamma (P less than or equal to .001), IL-1-alpha (P less than or equal to .001), and IL-2 (P less than or equal to .01) were found as compared to the patients with benign tumors and the controls, although total and differential leukocyte counts were similar in all three groups. A linear correlation between the levels of IFN-gamma and IL-1-alpha and the tumor stages could be shown. Our results suggest that a growing tumor burden may induce increasing immunological deficiencies as reflected by a decreasing cytokine production of the immune cells.


G test, a new direct method for diagnosis of Candida infection: comparison with assays for beta-glucan and mannan antigen in a rabbit model of systemic candidiasis.

An indirect method to measure beta-glucan, a major structural component of yeast cell walls, is available, but has the disadvantage of requiring the combined use of two assays. Recent reports describe the fungal index, which measures the difference between the conventional limulus test, in which factors C and G react with endotoxin and beta-glucan, and a new endotoxin-specific test, in which only factor C reacts with endotoxin. The G test was developed as a direct method to measure beta-glucan, and contains only factor G reacting with beta-glucan alone. In this study, the G test was examined in sera of rabbits with experimental systemic candidiasis, and compared with the fungal index and mannan assay. The G test showed positive in all rabbits with systemic candidiasis faster and with higher titers than with the fungal index. Three rabbits with fulminant systemic candidiasis showed higher levels of reactivity with the G test and the fungal index than two rabbits with mild reactions. Mannan was positive by at least one serum in four of five rabbits by the latex agglutination test, and there was a good correlation between these assays. The G test is a good serodiagnostic method for the detection of candidiasis.


Immunoblot assay for detection of autoantibodies in autoimmune disease.

When detecting antinuclear antibodies (ANA) a similar indirect immunofluorescence (IIF) pattern may be produced by antibodies which correspond to different antigenic specificity or to diverse autoimmune disorders. To circumvent this problem, we used an immunoblotting against total antigen from HEp-2 cells (TA-HEp-2-C) which proved to be more specific and sensitive than IIF, since it detected up to 40 antigenic bands and an immunoblot pattern characteristic of each individual and unrelated to the fluorescence patterns of ANA. The analysis of immunoblot patterns is of great diagnostic value, since in patients with systemic lupus erythematosus (SLE) the antigenic zone is found between 21 and 48 kDa, whereas in other rheumatic diseases with or without positive ANA antibodies by IIF it is found between 14 and 21 kDa. Bands which appear after 50 kDa may or may not have pathological significance since they are present in most normal individuals. The use of reference sera allowed the identification in the extract of previously described antigens relevant to these diseases. Some patients showed bands which have yet to be reported and may be clinically significant on their own or in association with other autoantibodies. The long-term follow-up of these immunoblot patterns may prove to be of prognostic importance.


Serum magnesium, copper, and zinc concentrations in acute myocardial infarction.

This study was carried out to assess the serum levels of magnesium, copper, and zinc after an acute episode of myocardial infarction. Determination of the metal concentrations were carried out on 41 patients with myocardial infarction and 41 healthy controls matched for age and sex. A slight decrease in the mean level of magnesium (P less than .05) was observed in patients (2.0 mg/dl) compared with the controls (2.1 mg/dl). The mean serum copper concentration was significantly higher (P less than .001) in patients (138 micrograms/dl) than in controls (98 micrograms/dl), while the mean serum zinc concentration was significantly lower (P less than .001) in patients (75 micrograms/dl) than in controls (100 micrograms/dl). The differences in serum copper and zinc levels between patients and controls were magnified considerably when the copper/zinc ratios were calculated for both groups (P less than .001). The mean copper/zinc value obtained for patients (1.91) was almost double that for the controls (1.02).


Monoclonal antibody-based ELISA to detect glycoprotein tumor-associated-antigen-specific immune complexes in cancer patients.

This report describes the development and applicability of a tumor-associated-antigen-specific immune complex (IC) detection assay to denote the presence of tumor cells in a cancer host. This assay utilizes a murine monoclonal antibody, AD1-40F4, which was produced to a glycoprotein tumor-associated antigen (TAA). In Western blot, the murine monoclonal antibody recognized a 90-100 kD subunit of the antigen. This antigen is immunogenic in cancer patients and induces formation of endogenous antigen-antibody complexes. Analysis of 250 sera from normal individuals and 419 sera from cancer patients revealed that a significantly (P less than .0005) greater proportion (234/419; 55.9%) of cancer sera was positive for the marker than the normal sera (8/250; 3.2%). The incidence of the 90 kD-TAA-specific-IC was consistently and significantly higher in melanoma (58.5%; 38/65), sarcoma (52.9%; 83/157), and carcinoma of breast (50.9%; 58/114), lung (68.2%; 30/44), and colon (64.1%, 25/39) than in the normal group. The age of serum donors did not affect the incidence of the reactivity. Also, at least in the cancer group the gender of the serum donor did not affect the incidence of the 90 kD-TAA-specific-IC positivity. In a retrospective study where sequential serum samples obtained postoperatively from 105 patients with melanoma were analyzed, the 90 kD-TAA-specific-IC could be detected in 72 (69%) of patients several years before the appearance of clinically detectable disease.(ABSTRACT TRUNCATED AT 250 WORDS)


The demonstration of a subset of carcinoid tumours of the appendix by in situ hybridization using synthetic probes to proglucagon mRNA.

Previous studies using immunohistochemistry have shown variable hormone production by carcinoid tumours of the appendix. In order to confirm the existence of a specific subset of these tumours, in situ hybridization using synthetic oligonucleotide probes to detect pre-proglucagon and pre-proinsulin mRNA was performed in formalin-fixed, paraffin-embedded material from eight tubular carcinoids, 12 insulin carcinoids, and two mucinous carcinoids. The results were correlated with standard silver and mucin stains. All tubular carcinoids but none of the insular or mucinous carcinoids contained proglucagon mRNA. Proinsulin mRNA was not detected in any of the tumours. Tubular carcinoids of the appendix constitute a definable subset of appendiceal carcinoids which have a similar distribution and prognosis to typical insular carcinoids and can be diagnosed on haematoxylin and eosin-stained sections confirmed by routine special stains. The main need for recognition is to avoid confusion with mucinous carcinoids, which have a worse prognosis and may require more aggressive treatment.


TDM35--a new monoclonal antibody to the XH1 cervical carcinoma cell line. Characterization and immunoperoxidase localization in benign and malignant tissues.

The murine monoclonal IgG1 kappa antibody TDM35 was raised against the cervical carcinoma cell line XH1. The antibody recognizes 18.5-66 kDa NCA-like glycoproteins and immunostains a variety of formalin-fixed, paraffin-embedded normal, benign, and malignant tissues. It is of value in the diagnosis of carcinoma of the exocrine pancreas and it identifies foci of squamous and glandular differentiation in other tumours. TDM35 should form a useful addition to a panel of antibodies for the evaluation of epithelial lesions.


Oral contraceptive use and histopathology of cancerous breasts in young women. Members of the U.K. National Case-Control Study Group.

A retrospective histopathological study of 300 women under 36 years of age was carried out to determine whether breast cancers occurring in oral contraceptive users showed any differences in pathological features compared with non-users. The patients belong to an age group in which an increased risk of cancer development has been reported following oral contraceptive usage. The incidence of non-neoplastic conditions in the residual breast was also studied in the two groups. There was little difference between breast cancers arising in pill users and non-users but in the residual non-neoplastic breast a decreased incidence of cysts and blunt duct adenosis was found in current users of the contraceptive pill. In contrast, lactational foci were found only in the breasts of pill users. The incidence of intraductal hyperplasia was not significantly different in the two groups.


Flow cytometric DNA-histogram analysis: non-stoichiometric fluorochrome binding and pseudoaneuploidy.

Detection of aneuploid subpopulations using flow cytometry requires stoichiometric binding of nucleic acid-specific fluorochromes onto DNA. It is shown that parameters like cell type specificity and differentiation stage, cell cycle stage, loss of DNA-integrity, cell preparation, and cytochemistry affect fluorochrome binding to DNA and give rise to the appearance of pseudo-aneuploid cell populations. Intercalating as well as non-intercalating fluorochromes show non-stoichiometric DNA-labelling in cell populations with identical DNA content, and pseudo-aneuploidy was found in flow cytometers equipped with either arc lamps or argon lasers. Pseudo-aneuploidy was never observed with intercalating and non-intercalating fluorochromes within identical specimens, consisting of cells of various differentiation states (e.g., bone marrow) or containing large numbers of dead cells. Therefore, fluorochromes exhibiting different base-pair specificities or steric binding modes should be applied to be sure of the correct interpretation of small levels of hypo- or hyper-diploidy (+/- 20 per cent).


Pathogenesis of pancreatic perilobular necrosis in patients with liver disease.

We often see perilobular necrosis of the pancreas in patients with liver disease at autopsy. This study was undertaken to determine the frequency and the mechanism of development of pancreatic perilobular necrosis in patients with liver disease. Pancreatic perilobular necrosis was seen in 21 per cent of 261 autopsied patients: in 41 per cent of 73 autopsied patients with liver disease and in 13 per cent of 188 autopsied patients without liver disease. Moreover, splanchnic congestion was present in 90 per cent of 30 pancreatic perilobular necrosis patients with liver disease. These data indicate that patients with liver disease develop perilobular necrosis of the pancreas more often than patients without liver disease, and that the high frequency may be a sequela of splanchnic congestion; that is, congestion of the pancreas and endotoxaemia due to congestion of the gut.


Collagen fibril structure of normal, aging, and osteoarthritic cartilage.

The collagen architecture in normal, aging, and osteoarthritic articular cartilage was studied optically using a new silver staining technique based on specimens from 50 autopsy cases, four amputated limbs, and six osteoarthritic knees. In the normal articular cartilage, the collagen fibrils in the superficial zone were compactly arranged into layers of decussating flat ribbons mostly parallel to the artificial split lines. The fibrils showed a tendency to condense into vertical arcade columns undergirded by tangential bundles in the intermediate zone. In the deep zone, the fibrils formed a random meshwork with a slight preponderance of vertical fibrils in the perilacunar region. Three types of early degradative lesions involving the collagen network were identified. Type I lesions consisted of focal superficial disruptions related to age and friction. Type II lesions consisted of focal disruptions of tangential fibrils in the intermediate zone leading to cyst formation, probably representing a form of local stress failure. Type III lesions were found in the patella and consisted of marked swelling of the superficial zone, the cause of which was unknown. Lesions of varying severity were seen within each of the three types; the morphological changes of the more severe lesions overlapped with those of clinically overt osteoarthritis.


Transient absorption spectra and quenching of coumarin excited states by nucleic acid bases.

Triplet-triplet absorption spectra of coumarin show different profiles and maxima in ethanol from those in water, which are very similar to those reported in benzene. Long-lived transient species other than triplet states were generated as initial photoproducts between coumarins and nucleic acid bases. The excited singlet and triplet states of coumarins were quenched by nucleic acid bases. Adenine base quenched the excited singlet state of coumarins more efficiently than thymine base. However, photocycloadducts of furocoumarins are formed predominantly with thymine, and not with adenine. Moreover, it is reported that the poly[dA-dT].poly[dA-dT] sequence region is the most favourable site for the photocycloaddition reaction of furocoumarins. The results imply that adenine contributes to singlet-state photocycloaddition reaction of furocoumarins with thymine, probably through an adenine-furocoumarin-thymine termolecular interaction.


Photochemical release of bases from nucleosides and their derivatives by water-soluble iron(III) porphyrins.

The compounds [meso-tetrakis(1-methyl-4-pyridiniumyl)porphyrinato]iron(III ) (FeIIITMPyP) and [meso-tetrakis(3,5-dichloro-1-methyl-4-pyridiniumyl)porphyrinat o]iron(III) (FeIIITCl2MPyP) photocatalysed the release of bases from nucleosides and their derivatives. The ribonucleosides, of which cytidine (C) gave the highest yield, produced much higher yields of free base than the corresponding deoxyribonucleosides. Under an argon atmosphere, virtually quantitative reduction of FeIIITMPyP into FeIITMPyP by C or 2'-deoxycytidine (dC) was observed and the reduction by C was much more effective than by dC. The increased reactivity of ribonucleosides relative to deoxyribonucleosides was ascribed to a difference in the binding properties of the porphyrin-nucleoside interactions and to base-releasing degradation of ribonucleosides from their C-2' carbon radical.


The laws of delayed photohaemolysis sensitized by chlorin e6.

The relationships between the rate of post-irradiated photohaemolysis sensitized by chlorin e6 and parameters such as the light fluence (time of irradiation) and sensitizer concentration were studied. On the basis of the single-parametric approach proposed by Valenzeno and Pooler, it was found that the haemolytic rate varies with the square of both the light fluence and the sensitizer concentration. Thus it can be concluded that, in a single erythrocyte lesion, two chlorin e6 molecules participate, each absorbing one photon. The possibility of suppression of post-irradiation haemolysis was also studied using the lipophilic antioxidant, butylated hydroxytoluene (BHT), and scavengers of 1O2, O2.- and HO. radicals. It was found that BHT inhibits, to a considerable extent, the post-irradiation lysis of cells, by about a factor of 2.5 at a BHT concentration of 9 microM. The addition to the medium of NaN3 (a scavenger of 1O2), superoxide dismutase (a scavenger of O2.- radicals), ethanol and D-mannitol (scavengers of HO. radicals), when irradiation was interrupted, did not produce a marked influence on the kinetics of subsequent haemolysis. On the basis of the results obtained, the nature of erythrocyte targets, which are crucial for the photodynamic effect of chlorin e6, is discussed.


Retinal photoisomerase: role in invertebrate visual cells.

In invertebrate visual cells, the rhodopsin content is maintained at a high level by the fast process of photoregeneration during daylight. Rhodopsin is converted by photoabsorption to metarhodopsin, which is reconverted to rhodopsin by light. In addition, rhodopsin is regenerated by a slow process of renewal which takes days to complete and involves the biosynthesis of opsin. It is well known that rhodopsin can be formed from opsin only when 11-cis-retinal is present; this requires the existence of an isomerizing enzyme which is capable of transforming all-trans-retinal, released from the degradation of metarhodopsin, into the 11-cis-retinal isomer. In some invertebrate visual systems, experiments on rhodopsin regeneration have been interpreted by assuming that the isomerization reaction is a light-dependent process involving a retinal-protein complex. Two retinal photoisomerases which have been well characterized, i.e. bee photoisomerase and cephalopod retinochrome, are reviewed here. Their properties are compared in order to determine their physiological role, which is likely to be in the renewal of visual pigment rhodopsin. To conclude, a visual pigment cycle is proposed in which rhodopsin regeneration follows two light-dependent pathways. This greatly simplifies the rhodopsin regeneration scheme for invertebrate visual systems.


Photodynamic activity of chlorin e6 and chlorin e6 ethylenediamide in vitro and in vivo.

Several parameters of chlorin e6 and its derivative chlorin e6 ethylenediamide have been investigated as these compound are potential sensitizers for photodynamic therapy. A study carried out to compare the cellular uptake of the pigments indicates that chlorin e6 ethylenediamide possesses an enhanced affinity for tumour cells and cellular membranes. Comparison of the uptake in induced sarcoma shows that chlorin e6 ethylenediamide is a much better tumour localizer than chlorin e6. The efficiency of phototherapy with chlorin e6 ethylenediamide is higher than that with chlorin e6. These data show the influence of the substitution of the carboxyl groups in chlorin e6 by ester and amide groups on the photosensitizing properties of the pigments.


Synergistic effects of photoactivated tetra(4-sulfonatophenyl)porphine and nocodazole on microtubule assembly, accumulation of cells in mitosis and cell survival.

Human carcinoma cells of the line NHIK 3025 were incubated with meso-tetra(4-sulfonatophenyl)porphine (TPPS4) for 18 h and exposed to light in the absence or presence of nocodazole. Nocodazole (1 microgram ml-1) was applied to the cells 15 min prior to light exposure and washed off the cells immediately afterwards. The presence of nocodazole during photoactivation of TPPS4-loaded cells leads to a significantly reduced ability of tubulin to repolymerize after withdrawal of nocodazole, an increased accumulation of the cells in mitosis with a larger fraction in c-metaphase and a higher yield of photoactivated cells. A higher proportion of the cells accumulating in mitosis 6-12 h after exposure to light is unable to form colonies when exposed to light in the presence of nocodazole than in its absence. The present results are consistent with a specific TPPS4-induced photodamage to the unpolymerized form of the microtubule components.


Phototoxicity of non-steroidal anti-inflammatory drugs: in vitro testing of the photoproducts of Butibufen and Flurbiprofen.

In this work, the phototoxicity of two non-steroidal anti-inflammatory drugs, Butibufen and Flurbiprofen, was examined. Both were unstable to light, to give several photoproducts which were isolated and identified. The different photoproducts were formed by a primary photochemical mechanism which involves an initial cleavage of the C-C bond alpha to the carbonyl group, followed by several secondary processes. The cytotoxic effects of the xenobiotics were evaluated using two well-established biological in vitro tests: (a) enzyme leakage lactate dehydrogenase and glutamate-oxaloacetate transaminase from cultured fibroblasts and (b) lysis of red blood cells. The benzylic alcohols caused extensive leakage from cultured fibroblasts at the different concentrations assayed. The alcohol obtained from Butibufen was a potent lytic agent for human red blood cells. The other photoproducts, Butibufen and Flurbiprofen did not produce observable toxic effects on cells.


Endogenous protoporphyrin IX, a clinically useful photosensitizer for photodynamic therapy.

The tissue photosensitizer protoporphyrin IX (PpIX) is an immediate precursor of heme in the biosynthetic pathway for heme. In certain types of cells and tissues, the rate of synthesis of PpIX is determined by the rate of synthesis of 5-aminolevulinic acid (ALA), which in turn is regulated via a feedback control mechanism governed by the concentration of free heme. The presence of exogenous ALA bypasses the feedback control, and thus may induce the intracellular accumulation of photosensitizing concentrations of PpIX. However, this occurs only in certain types of cells and tissues. The resulting tissue-specific photosensitization provides a basis for using ALA-induced PpIX for photodynamic therapy. The topical application of ALA to certain malignant and non-malignant lesions of the skin can induce a clinically useful degree of lesion-specific photosensitization. Superficial basal cell carcinomas showed a complete response rate of approximately 79% following a single exposure to light. Recent preclinical studies in experimental animals and human volunteers indicate that ALA can induce a localized tissue-specific photosensitization if administered by intradermal injection. A generalized but still quite tissue-specific photosensitization may be induced if ALA is administered by either subcutaneous or intraperitoneal injection or by mouth. This opens the possibility of using ALA-induced PpIX to treat tumors that are too thick or that lie too deep to be accessible to either topical or locally injected ALA.


Dark induction of haem oxygenase messenger RNA by haematoporphyrin derivative and zinc phthalocyanine; agents for photodynamic therapy.

Haematoporphyrin derivative is one of the main drugs currently used in clinical trials involving photodynamic therapy of cancer, and zinc phthalocyanine is being considered as one of several possible alternatives. We show that incubation of cultured human fibroblasts populations with either of the two drugs will lead to a sharp increase in the accumulation of the messenger RNA corresponding to haem oxygenase. Only cells incubated with haematoporphyrin derivative show additional enhancement of expression of this specific gene on exposure to red light. Since haem oxygenase induction appears to be a specific stress response that may be involved in cellular defence, such observations should be confirmed under conditions which would allow the clinical implications to be fully evaluated.


Excited singlet state properties of anthracenedione photosensitizers.

The absorption, fluorescence and S1 state kinetics of anthracycline antitumour drugs (e.g. daunomycin, adriamycin) and several imino- and/or amino-substituted derivatives are investigated. The study, which includes all anthracyclines which possess photocytocidal activity, is extended to the disubstituted aminoanthracenedione, mitoxantrone, a red-light-absorbing antitumour drug whose activity, both in vitro and in vivo, is enhanced by photoactivation. The S1 state of the anthracycline imino and amino derivatives, in aqueous buffer at pH 7.4, is characterized by bi-exponential decay kinetics which indicates the presence of two ground state populations differing in the extent of hydrogen bonding. The ammonium group of the sugar moiety of anthracyclines contributes to the quenching of the S1 state population through a prototropic mechanism.


Ferric-ion-photosensitized damage to DNA by hydroxyl and non-hydroxyl radical mechanisms.

Iron(III) and UVA (320-400 nm) light strongly diminished the transforming activity of Haemophilus influenzae DNA in the presence of oxygen. Iron(III) alone in the absence of light had no measurable effect on the transforming activity. The chelating agent ethylenediaminetetraacetic acid (EDTA) conferred virtually complete protection, but hydroxyl radical scavengers (mannitol, methanol, ethanol, isopropanol and dimethyl sulfoxide) inhibited only a small fraction of the inactivation. Treatment of plasmid DNA (pBR322) with iron(III) results in the conversion of the covalently closed circular form of the plasmid to open circles and ultimately to the linear form. Concomitant with the alteration in the conformation of the plasmid, the ability to transform Escherichia coli was reduced. In model systems, iron(III) photoreacted with the DNA backbone causing nicking and double-strand breakage. The results are consistent with a mechanism involving a preliminary complexation of iron(III) by DNA followed by the generation of reactive free radicals other than .OH. We suggest that bound iron, or other UV-absorbing transition metal complexes, may be chromophores capable of causing DNA damage in the long-wave near-UV region.


Ultrastructural damage in photosensitized endothelial cells: dependence on hematoporphyrin delivery pathways.

The subcellular photodamage to endothelial cells in culture, revealed by transmission electron microscopy, was correlated with discrete delivery pathways of hematoporphyrin (HP). Cell detachment from the extracellular matrix, prominent water influx starting at the outer membrane and formation of blebs followed by cell death were the result of photodynamic damage induced by aqueous HP. Serum-bound HP was internalized by endocytosis and accumulated in lysosomal compartments as located after photosensitization. Obstructed lysosomal membranes, degradation of chromatin and swelling of endoplasmic reticulum were revealed in these cells. Red blood cells (RBCs), preincubated with HP, delivered low amounts of the drug to endothelial cells. The photodamage was limited to the nucleus and nucleolus. The role of photosensitizer delivery pathways in cancer cell damage is discussed.


Mode of photocatalytic bactericidal action of powdered semiconductor TiO2 on mutans streptococci.

Powdered semiconductor TiO2 has a photocatalytic bactericidal capacity on some kinds of bacteria, but its mechanism still remains unclear. The mode of its photocatalytic bactericidal action on the mutans group of streptococci was investigated. Powdered TiO2 had a bactericidal capacity on all serotypes of mutans streptococci. Streptococcus sobrinus AHT was mainly used for these experiments. The most effective concentration of TiO2 was about 1 mg ml-1 and, at this concentration, 10(5) colony-forming units of S. sobrinus AHT per millilitre were completely killed within 1 min. In order to search for the mechanism of this effect, a high bacterial cell density (10(9) colony-forming units ml-1) was used in the following studies. "Rapid" leakage of potassium ions from the bacteria occurred parallel to the decrease in cell viability. Protein and RNA were "slowly" released from bacterial cells for a reaction time up to 120 min. The pH of the reaction mixture decreased continuously to 4.5 after 120 min. Co-aggregation of S. sobrinus AHT and powdered TiO2 occurred at high bacterial densities (above 10(8) colony-forming units ml-1). Aggregates gradually decomposed with light irradiation. Transmission electron microscopy of S. sobrinus AHT after photocatalytic action for 60-120 min indicated complete destruction of bacterial cells. From these results, bacterial death appears to be caused by a significant disorder in cell membranes and finally the cell walls were decomposed.


Laryngotracheobronchitis as a complication of measles during an urban epidemic.

To evaluate demographic and clinical correlates of laryngotracheobronchitis (LTB) as a complication of measles during a community-wide epidemic. Retrospective review of medical records. Childrens Hospital Los Angeles, a large urban pediatric facility, during a regional epidemic of measles studied January through June 1990. All patients identified at our hospital who met Centers for Disease Control criteria for measles. Of 440 patients with measles, 82 also had LTB (18.6%). Patients in whom LTB developed were significantly younger (mean +/- SD: 14.7 +/- 8.2 months) than the cohort (24.8 +/- 30.1 months) (p less than 0.001) and more likely to require hospitalization (91.5%) than the cohort (44.3%) (p less than 0.001). Thirteen patients (17.3%) required intensive care, including 9 (11%) who required endotracheal intubation for a mean of 8.3 +/- 7.1 days. Pulmonary function testing of five patients with an endotracheal tube in place, including three not clinically assessed as having pneumonia, indicated the presence of concomitant lower respiratory tract disease. Laryngotracheobronchitis was a frequent and often severe complication of measles. The likelihood that LTB would develop was inversely related to age, generally required inpatient care, and necessitated endotracheal intubation in severely affected patients.


Role of immunoglobulin subclasses and specific antibody determinations in the evaluation of recurrent infection in children.

We studied humoral immune function in 267 children with recurrent respiratory infections referred to our immunology clinic to determine the most appropriate immunologic studies for evaluating recurrent infections in children. Of this highly selected population, 58% had a partial deficiency in one or more of the major immunoglobulin isotypes or IgG subclasses (defined as at least 2 SD below the normal age-adjusted mean). In none of the patients was there a total absence of an immunoglobulin isotype. The most common abnormality was partial IgA deficiency, which was found in one third of the patients. Twenty-six patients had only partial IgG subclass deficiencies, of which 20 were deficiencies of a single subclass. IgG1 was an isolated partial defect in three patients, IgG3 in five patients, and IgG2 and IgG4 were selective partial defects in six patients each. Tetanus toxoid and pneumopolysaccharide type 3 were the most immunogenic of the immunogens tested; hyporesponsiveness to pneumococcal polysaccharide types 7, 9, and 14 was common. Nineteen percent of the patients with normal immunoglobulin concentrations who were tested had lower-than-expected antibody titers; 42% of those tested with partial isotype deficiencies had deficient antibody responses. Of 25 patients with selective partial IgG subclass deficiencies or combined IgG subclass deficiencies, eight had antibody deficiencies. Our findings indicate that a high proportion of children referred to immunology clinics for recurrent infection have a demonstrable immunologic abnormality. Selective IgG subclass deficiency or a combined IgG subclass deficiency without an associated deficiency in a major immunoglobulin isotype is unusual. Identification of such patients is not predictive of the capacity to form antibodies to the antigens tested in this study and, in our opinion, adds little to the initial evaluation of immune function in such children.


Antibody response to Bordetella pertussis antigens after immunization with American and Canadian whole-cell vaccines.

Because of apparent differences in the incidence and epidemiology of pertussis in the United States and Canada, we measured the antibody response to four Bordetella pertussis antigens and to a whole-bacteria preparation in children immunized with American and Canadian whole-cell pertussis vaccines. All infants received combined pertussis, tetanus, and diphtheria vaccines from one of two American manufacturers or a single Canadian manufacturer. The Canadian children received either oral poliomyelitis vaccine, inactivated poliomyelitis vaccine as a separate injection, or a product that combined inactivated poliomyelitis vaccine with diphtheria, tetanus, and pertussis components. The Canadian trivalent diphtheria, tetanus, and pertussis vaccine given with oral poliovirus vaccine induced lower anti-pertussis toxin antibody titers than did the American vaccines (p < or = to 0.05) but higher antifimbriae and anti-69-kilodalton outer-membrane protein (pertactin) antibody titers (p < or = to 0.02). Canadian children immunized with inactivated poliomyelitis vaccine either as a separate injection or as a combined diphtheria, tetanus, and pertussis vaccine had consistently lower pertussis antibody titers than did those who received oral poliomyelitis vaccine (p < or = 0.001). We conclude that there is a wide range of antibody responses to B. pertussis antigens after immunization with various whole-cell pertussis vaccines, and that these responses may be influenced by concurrent administration of other vaccines.


Evaluation of gonadotropin responses to synthetic gonadotropin-releasing hormone in girls with idiopathic hypopituitarism.

We hypothesized that prepubertal girls with gonadotropin deficiency would produce less follicle-stimulating hormone (FSH) in response to synthetic gonadotropin-releasing hormone (GnRH) than would gonadotropin-sufficient children. To test this hypothesis, we performed 103 GnRH tests serially in 21 children who had idiopathic hypopituitarism with growth hormone deficiency. We tried to predict whether puberty would occur in the 17 girls with bone ages of 8 years or less. Of these 17 girls, 4 failed to have spontaneous secondary sexual characteristics by age 16 1/2 years, and 12 had spontaneous complete pubertal development. One girl had incomplete pubertal maturation with partial gonadotropin deficiency; her results were combined with those of the girls who had no spontaneous pubertal development. With increasing bone age, the girls with complete pubertal development had a decrease in the increment of FSH released in response to GnRH, although basal gonadotropin concentrations did not change. For GnRH tests performed at bone ages of 8 years or less, basal luteinizing hormone (LH) values did not differ between girls with complete puberty and those with absent or incomplete puberty. However, basal FSH and the incremental response of LH and FSH to GnRH were greater in those with complete puberty. Only two girls with prepubertal bone ages at the time of testing, who subsequently had complete puberty, had incremental FSH responses to GnRH that were less than 5 IU/L. Individual incremental LH responses to GnRH did not discriminate well between groups. None of the girls with adrenocorticotropic hormone deficiency, either originally or subsequently, had spontaneous puberty, but 4 of 12 girls with thyrotropin deficiency, either originally or subsequently, had complete puberty. We conclude that a significant increase in GnRH-stimulated FSH suggests that spontaneous pubertal development will occur in girls with idiopathic hypopituitarism. However, a low FSH response to GnRH may not be diagnostic of gonadotropin deficiency.


Thiamine, riboflavin, and pyridoxine deficiencies in a population of critically ill children.

The unexpected autopsy finding of Wernicke encephalopathy in three children who died after prolonged enteral feeding prompted us to examine the incidence of thiamine deficiency in three high-risk pediatric populations. We also measured riboflavin and pyridoxine activity in the same groups. We used activated enzyme assays (erythrocyte transketolase, glutathione reductase, aspartate aminotransferase) to assess tissue stores of the dependent vitamin cofactors (thiamine (vitamin B1), riboflavin (vitamin B2), and pyridoxine (vitamin B6), respectively). Using our own reference ranges based on data from 80 healthy adults and children, we prospectively investigated the B vitamin status of three groups of children: (1) 27 patients who were fed solely by nasogastric tube for more than 6 months, (2) 80 children admitted to a pediatric intensive care unit for more than 2 weeks, and (3) 6 children receiving intensive chemotherapy. The upper limits for stimulated enzyme activity in control subjects were unaffected by age or gender (16% for transketolase, 63% for glutathione reductase, 123% for aspartate aminotransferase). Using these limits, 10 (12.5%) of 80 patients receiving intensive care and 4 of 6 patients receiving chemotherapy were thiamine deficient. Elevated levels returned to normal after thiamine supplementation. No patients were pyridoxine deficient, but 3 (3.8%) of the 80 patients receiving intensive care and 1 of the 6 patients receiving chemotherapy were also riboflavin deficient. We conclude that unrecognized thiamine deficiency is common in our pediatric intensive care and oncology groups. This potentially fatal but treatable disease can occur in malnourished patients of any age and is probably underdiagnosed among chronically ill children. Our findings may be applicable to other high-risk pediatric groups.


Communicating medical bad news: parents' experiences and preferences.

Parents (N = 189) of children enrolled in 15 developmental day care centers completed questionnaires that examined the experience of being told bad news and elicited preferences for physician behavior in a hypothetical situation (communicating the diagnosis of Down syndrome). Parents, in comparison with their experiences, preferred (p < 0.001) more communication of information and feelings by their physician. Their strongest preferences were for physicians to show caring (97%), to allow parents to talk (95%), and to allow parents to show their own feelings (93%). They wanted physicians to share information (90%) and to be highly confident (89%). Most parents (87%) desired parent-to-parent referral, but only a few (19%) were referred. We conclude that there is a difference between what parents experience and what they desire in physicians who communicate bad news. Physicians control the interaction and are highly confident, but parents especially value physicians who show caring and allow parents to talk and share their feelings.


Participation in biomedical research: the consent process as viewed by children, adolescents, young adults, and physicians.

We examined the capacity of children, adolescents, and young adults to assent and consent to participation in biomedical research, and what physician-investigators believe is important for patients in these age groups to know about such participation. The sample included 44 male and female subjects, ranging in age from 7 to 20 years, who were hospitalized to treat either pediatric cancer or obesity. The participants completed a structured interview that assessed knowledge of research participation using the elements outlined in the federal guidelines for informed consent. The study subjects were most knowledgeable about those elements of consent that assessed concrete information (e.g., freedom to ask questions, time elements involved, and the benefits of participation). They were less knowledgeable about those elements of informed consent that assessed abstract information (e.g., scientific vs therapeutic purpose of the study, and alternative treatments). Chronologic age was not related to knowledge of the elements of informed consent. The strategies that the study subjects used to reason about participation in research appeared to parallel their reasoning about other physical phenomena.


Atypical features of the hepatic form of carnitine palmitoyltransferase deficiency in a Hutterite family.

We describe hepatic carnitine palmitoyltransferase (CPT I) deficiency in three children (a brother and sister and their second cousin) from an extended inbred Hutterite kindred. The patients were first seen between 8 and 18 months of age with recurrent episodes of hypoketotic hypoglycemia accompanied by a decreased level of consciousness and hepatomegaly. One patient had two Reye syndrome-like episodes. Abnormal organic acids were rarely detected in urine. Serum total and free carnitine levels were elevated in all three patients. Fibroblast acyl-coenzyme A dehydrogenase activities were normal in all, but palmitic acid oxidation, performed in fibroblasts from one patient, was less than 10% of control values. Activity of CPT I in cultured skin fibroblasts from the three patients was 10% to 15% of control levels; CPT II activity was normal. Activity of CPT I and CPT II in muscle from one patient was normal. Atypical features in two of these patients were greatly elevated levels of liver enzymes and creatine kinase during acute episodes. The patients have recently been successfully treated with medium-chain triglycerides and avoidance of fasting. Early identification and treatment of this disorder may avert potentially fatal episodes of hypoglycemia.


Initial observations of human dermatosparaxis: Ehlers-Danlos syndrome type VIIC.

We describe initial observations of an infant with dermatosparaxis (another form of Ehlers-Danlos syndrome, designated as type VIIC), an autosomal recessive disorder characterized by skin fragility and described in several species of domesticated animals. Electron microscopic examination of the skin shows collagen sheets rather than fibrils, and characteristic distortions resembling hieroglyphs. In addition to skin fragility, the disorder is characterized by redundant skin folds and edema, healing with minimal scar formation, large fontanels and wide sagittal and metopic sutures, blue sclerae, micrognathia, and umbilical hernia; after the neonatal period there are joint laxity, growth failure, short limbs, and normal mineralization of the skeleton except for the cranial vault. This disorder may also be a cause of premature rupture of placental membranes and myopia.


Prevention of rheumatic fever in Costa Rica.

During the beginning of the 1970s, major changes occurred in Costa Rica in the treatment of streptococcal throat infections. Because of poor compliance with regimens using orally administered agents, intramuscular administration of benzathine penicillin was selected as the standard treatment and throat cultures were eliminated as a prerequisite for prescribing antibiotics. A decline in the incidence of rheumatic fever then occurred. We believe that similar health intervention could be applied in other developing countries.


Chronic hepatitis B in adopted Romanian children.

Four of five Romanian orphans adopted by U.S. families were found to have chronic hepatitis B virus (HBV) infection after negative test results were reported in Romania before adoption. Another child with known HBV infection was found to be coinfected with hepatitis D virus. There is a high incidence of HBV infection in Romanian orphans, and results of tests for HBV are unreliable in Romania.


Bacillary angiomatosis in a child undergoing chemotherapy.

Bacillary angiomatosis is an infectious disease of the skin and viscera characterized by vascular lesions, originally described in patients with human immunodeficiency virus infection. There are also case reports of bacillary angiomatosis occurring in immunocompetent patients and in noninfected patients with suppressed immune function. We report a case of bacillary angiomatosis in a child undergoing chemotherapy for acute leukemia.


Erythrocyte macrocytosis in infants and children with Down syndrome.

Erythrocyte mean corpuscular volume and mean corpuscular hemoglobin levels were higher in children with Down syndrome than in normal control subjects. Reference values for mean corpuscular volume and mean corpuscular hemoglobin level derived from normal populations may be inappropriate for children with Down syndrome. These findings may have important implications for the diagnosis of iron deficiency in these children.


Intellectual development at age 12 years of children with congenital hypothyroidism diagnosed by neonatal screening.

Twenty-seven patients with congenital hypothyroidism diagnosed by neonatal screening were examined at the age of 12 years. The 12 patients with severe hypothyroidism at diagnosis (thyroxine < 2 micrograms/dl (< 26 nmol/L), and area-of-the-knee epiphyses < 0.05 cm2 had a lower IQ than the 15 patients with less severe hypothyroidism (mean +/- SD, 89 +/- 17 vs 104 +/- 10; p < 0.007). Comparisons of patients and siblings confirm that this difference was due to the severity of hypothyroidism.


Emergence of optic pathway gliomas in children with neurofibromatosis type 1 after normal neuroimaging results.

We report the appearance of gliomas of the optic nerve or chiasm in four young children with neurofibromatosis type 1 whose previous neuroimaging studies showed no abnormalities; the age range of the children was 1 year 8 months to 5 years 9 months at the time the tumors were detected. Normal neuroimaging findings in an infant or young child with neurofibromatosis type 1 does not provide assurance that the optic nerves and chiasm will remain healthy.


Marrow transplantation for thrombocytopenia-absent radii syndrome.

A two-year-old girl with thrombocytopenia-absent radii syndrome underwent transplantation of allogeneic bone marrow from her histocompatible sibling to correct her persistently low platelet count. Six years after transplantation, she has durable engraftment of allogeneic marrow and a normal platelet count that will allow her to undergo necessary corrective orthopedic procedures.


Effectiveness of dexamethasone in preventing extubation failure in preterm infants at increased risk for airway edema.

We studied 50 preterm infants who had multiple or traumatic endotracheal intubations, or whose duration of endotracheal intubation was > or = to 14 days, and who were considered at high risk for airway edema. These infants were enrolled in a prospective, randomized, controlled clinical trial to assess whether prophylactic dexamethasone therapy would be effective in the prevention of postextubation stridor and respiratory distress. At study entry, both groups had similar weights, postnatal ages, methylxanthine use, ventilator settings, blood gas values, and pulmonary function test results (dynamic compliance, total respiratory resistance, tidal volume, peak-to-peak transpulmonary pressure, minute ventilation, and peak inspiratory and expiratory flow rates). Patients underwent blood gas studies, physical examinations, and pulmonary function testing at baseline (4 hours before extubation) and again 2 to 4 hours and 18 to 24 hours after extubation. Twenty-seven infants received dexamethasone, 0.25 mg/kg per dose, at baseline, and then every 8 hours for a total of three doses; 23 infants received saline solution at corresponding times. Eighteen to twenty-four hours after extubation, total pulmonary resistance increased by 225% from baseline in the control group compared with 33% in the dexamethasone group (p < 0.006), and the dexamethasone group had a greater tidal volume, a greater dynamic compliance, and a lower arterial carbon dioxide pressure. Of 23 control infants, 10 had postextubation stridor compared with 2 of 27 dexamethasone-treated patients (p < 0.006). Of the 23 control patients, 4 required reintubation compared with none of the treated group (p < 0.05). We conclude that the prophylactic use of corticosteroids for the prevention of postextubation stridor and respiratory distress is efficacious in low birth weight, high-risk preterm infants.


Effect of dexamethasone therapy on fibronectin and albumin levels in lung secretions of infants with bronchopulmonary dysplasia.

The influence of dexamethasone on levels of total fibronectin (tFn), cellular fibronectin (cFn), plasma fibronectin (pFn), and albumin in lung secretions was determined in tracheal aspirate samples collected from 45 infants with bronchopulmonary dysplasia during a 6-week course of dexamethasone therapy. Secretory component for IgA (SC) was used as a reference protein. Thirty-seven infants (82%) survived and had their endotracheal tubes successfully removed. Corticosteroid therapy was associated with a significant decrease in the cFn/SC ratio. There was also a significant decrease in albumin/SC and pFn/SC ratios, suggesting decreased capillary permeability with corticosteroid therapy. Four of the remaining infants did not improve while receiving corticosteroids and died of respiratory failure at 3 to 8 weeks of age. In these "no response" infants, tFn/SC, cFn/SC, pFn/SC, and albumin/SC ratios when corticosteroid therapy was initiated were threefold to fourfold greater (p < 0.01) than ratios in survivors. Another group of four infants initially responded to corticosteroids but subsequently died with severe pulmonary cystic degeneration at 4 to 6 months of age; in these infants, tracheal aspirate tFn/SC, cFn/SC, and albumin/SC ratios were significantly lower than in survivors and remained unchanged during corticosteroid therapy. The decrease in the concentrations of plasma fibronectin and albumin in tracheal aspirate samples from the survivors suggests that the rapid clinical improvement seen in infants with bronchopulmonary dysplasia after the initiation of dexamethasone therapy is due in part to improvement in the integrity of the alveolar-capillary barrier. In addition, the decrease in the aspirate levels of cFn suggests the potential for corticosteroids to limit pulmonary fibrosis in the surviving infants. The depressed levels of fibronectin observed in the infants with severe cystic lung disease may represent an impaired healing response to lung injury.


Clinical and pharmacologic study of fetal supraventricular tachyarrhythmias.

The purpose of this study was to evaluate the efficacy of maternal digoxin administration in 16 cases of fetal supraventricular tachyarrhythmia diagnosed by fetal echocardiography; cardiac anatomy was normal in all cases. The retrospective analysis included nine mothers who received digoxin orally in most cases, with control of the arrhythmia in two fetuses. The addition of amiodarone (five cases) and propranolol (two cases) yielded two successes with amiodarone. The therapeutic regimen of digoxin was then modified on the basis of poor response to orally administered digoxin. In the prospective study, digoxin was administered intravenously to seven mothers according to a standard protocol; high doses (1 to 2 mg intravenously) were prescribed for the first 24 hours and intravenous digoxin therapy was maintained for at least 5 days, depending on the fetal response. Digoxin pharmacokinetic studies of four mothers showed an increased plasma clearance and reduced elimination half-life. Digoxin controlled the five supraventricular tachycardias (with hydrops in four cases). Maternal flecainide therapy restored sinus rhythm in two cases of atrial flutter. Our prospective study emphasizes the efficacy and safety for the fetus and the mother of intravenously administered digoxin as a first-choice drug in the treatment of supraventricular tachyarrhythmias. Flecainide may be a promising second-choice drug but requires further clinical investigation. Amiodarone and propranolol seem to be ineffective.


Clinical events in association with timing of intraventricular hemorrhage in preterm infants.

To ascertain whether any routine practices or clinical manipulations in a neonatal intensive care unit could induce intraventricular hemorrhage (IVH) in preterm infants, we performed ultrasonic monitoring of the germinal layer continuously for 48 hours in 33 extremely premature infants with respiratory distress. Intraventricular hemorrhage developed in 16 of these infants. In four infants the timing of the germinal layer hemorrhage was confirmed with ultrasonic monitoring. Three of the four cases were apparently associated with clinical events occurring at the moment of IVH: manual ventilation for improvement of hypercapnia associated with primary pulmonary hypertension of the newborn; correction of hyperkalemia, which was causing an arrhythmia, with administration of calcium gluconate and sodium bicarbonate; and administration of surfactant-TA to improve respiratory failure caused by pulmonary hemorrhage. In these three infants it appeared that one of the basic factors inducing IVH might be an increase in blood pressure with or without hypercapnia, causing cerebral reperfusion after ischemic damage of the germinal layer.


Neurologic sequelae in transient nonketotic hyperglycinemia of the neonate.

We report a neonate with the transient form of nonketotic hyperglycinemia manifested by extreme hypotonia, lethargy, apnea, and myoclonic and generalized convulsions in early neonatal life. Despite normalization of the biochemical values, severe neurologic sequelae were observed. This case suggests that the transient form of nonketotic hyperglycinemia sometimes causes severe brain damage.


Two-year results of treatment with depot leuprolide acetate for central precocious puberty.

We report results from 2 years of therapy with the long-acting form of the gonadotropin-releasing hormone (GnRH) analog leuprolide acetate, which was previously reported in short-term trials to be efficacious in the treatment of central precocious puberty. Thirteen girls and two boys, aged 1.9 to 9.7 years, who satisfied clinical criteria including GnRH-stimulated luteinizing hormone (LH) greater than 10 IU/L (mean radioimmunoassay LH, 29.1 +/- 5.54 IU/L), received depot leuprolide, 6 to 15 mg intramuscularly every 4 weeks. Estradiol (or testosterone), insulin-like growth factor I, and GnRH-stimulated gonadotropins were obtained at baseline, at 2 months, and at 6-month intervals with bone age determinations. Pubertal progression ceased in all patients, and menses did not occur. Mean increase in height during therapy was 5.77 +/- 2.0 cm/yr. Predicted adult height increased over baseline by 5.52 +/- 1.16 cm at 18 months. Mean estradiol values in the girls declined from 3.3 +/- 0.6 to 0.60 +/- 0.03 ng/dl, with no overlap of baseline and treatment values. The mean basal LH value was unchanged by therapy; mean basal and peak LH values for all follow-up GnRH stimulation tests were 4.05 +/- 0.57 and 4.95 +/- 0.70 IU/L, respectively. Basal and peak follicle-stimulating hormone (FSH) values were suppressed from 4.10 +/- 0.62 and 10.06 +/- 1.34 IU/L, respectively, to generally undetectable levels (< 1). Comparison with untreated control patients suggested that basal LH did not completely return to prepubertal levels, whereas FSH levels were suppressed below prepubertal levels. Estradiol, FSH, and LH levels reached their nadir by 2 months; in contrast, mean serum levels of insulin-like growth factor I progressively declined from +0.57 +/- 0.19 SD score to -0.06 +/- 0.22 SD score at 24 months. Two girls were withdrawn from the study because of reactions at injection sites, with apparent sterile abscess formation in one patient. This study provides evidence that (1) long-term treatment with depot leuprolide is characterized by immediate and sustained laboratory and clinical suppression, (2) GnRH-stimulated LH and random FSH and estradiol concentrations are useful laboratory measures of efficacy, and (3) the progressive increase in predicted adult height is temporally associated with decreased serum levels of insulin-like growth factor I and striking deceleration of bone age advancement.


Stimulation of statural growth by recombinant insulin-like growth factor I in a child with growth hormone insensitivity syndrome (Laron type)

We studied the effects of 9 months of treatment with twice-daily subcutaneous injections of insulin-like growth factor I (IGF-I), 120 micrograms/kg per dose, in a 9.7-year-old child with growth hormone insensitivity syndrome, in whom short-term studies had suggested that IGF-I might promote linear growth. Height velocity increased from 6.5 cm/yr (+1.7 SD score) to 11.4 cm/yr (+8.8 SD score). Serum concentrations of IGF-I increased from pretreatment values of 9 +/- 2 micrograms/L to a peak of 347 +/- 26 micrograms/L after 2 hours. Serum concentrations of IGF-II were unchanged. Basal but not stimulated growth hormone concentrations were decreased. During the first 12 days of treatment, serum concentrations and the 24-hour urinary excretion of urea nitrogen were decreased by 28% and 10%, respectively (p < 0.05), there was a 2.4-fold increase in urinary excretion of calcium (p < 0.001), and creatinine clearance and urine volume increased by 22% and 55%, respectively (p < 0.02). The changes in serum levels of urea nitrogen and in urinary calcium and creatinine clearance were still evident at 10 weeks. Fasting and postprandial serum glucose concentrations remained normal. We conclude that IGF-I given as twice-daily subcutaneous injections is effective in stimulating statural growth without producing the hypoglycemia and hyperglycemia observed when IGF-I is infused continuously.


Use of analgesic agents for invasive medical procedures in pediatric and neonatal intensive care units.

The purpose of this study was to assess the use of analgesic agents for invasive medical procedures in pediatric and neonatal intensive care units. The directors of 38 pediatric units and 31 neonatal units reported that analgesics were infrequently used for intravenous cannulation (10%), suprapubic bladder aspiration (8%), urethral catheterization (2%), or venipuncture (2%). Analgesics were used significantly more regularly in pediatric than in neonatal intensive care units for arterial line placement, bone marrow aspiration, central line placement, chest tube insertion, paracentesis, and lumbar puncture.


Myalgia and elevated creatine kinase activity associated with subcutaneous injections of diluent.

A 16-year-old boy with short stature and mild primary hypothyroidism received subcutaneous injections of either recombinant human growth hormone or placebo in diluent that contained glycerol and m-cresol as a preservative. While he was receiving the study drug, myalgia developed and serum creatine kinase values were elevated. Both resolved when injections were stopped and recurred when injections of diluent alone were given. The myalgia and elevated creatine kinase activity were apparently caused by a component of the diluent.


Energy-linked mitochondrial pyridine nucleotide transhydrogenase of adult Hymenolepis diminuta.

Employing "phosphorylating" submitochondrial particles as the source of pyridine nucleotide transhydrogenase, the occurrence of an energy-linked NADH----NADP+ transhydrogenation in the adult cestode Hymenolepis diminuta was demonstrated. The isolated particles displayed rotenone-sensitive NADH utilization and the reversible transhydrogenase, with the NADPH----NAD+ transhydrogenation being more prominent. Although not inhibiting the NADPH----NAD+ reaction, rotenone, but not oligomycin, inhibited the catalysis of NADH----NADP+ transhydrogenation. In the presence of rotenone, Mg2+ plus ATP stimulated by more than 3-fold NADH----NADP+ transhydrogenation. This stimulation was ATP specific and was abolished by EDTA or oligomycin. Succinate was essentially without effect on the NADH----NADP+ reaction. These data demonstrate the occurrence of an energy-linked transhydrogenation between NADH and NADP+ with energization resulting from either electron transport-dependent NADH oxidation or ATP utilization via the phosphorylating mechanism in accord with the preparation of "phosphorylating" particles. This is the first demonstration of an energy-linked transhydrogenation in the parasitic helminths and apparently in the invertebrates generally.


Isoenzyme analysis of 35 Toxoplasma gondii isolates and the biological and epidemiological implications.

Isoenzyme analysis was conducted on the tachyzoite stage of 35 Toxoplasma gondii isolates. Fifteen enzyme systems were studied after isoelectrofocusing of tachyzoite extracts in polyacrylamide or agarose gels. Six enzyme systems showed variable electrophoretic patterns: aspartate aminotransferase (EC 2.6.1.1), glutathione reductase (EC 1.6.4.2), glucose phosphate isomerase (EC 5.3.1.9), amylase (EC 3.2.1.1), acid phosphatase (EC 3.1.3.2), and propionyl esterase. Their combination allows the description of 5 zymodemes among the 35 T. gondii isolates. Zymodeme 1 involves 6 isolates that are highly pathogenic to mice and for which oocysts could not be obtained. Isolates belonging to zymodemes 2, 3, and 4 are less pathogenic to mice and produced oocysts. Zymodeme 5 involves only 1 isolate, which was highly pathogenic to mice.


A new species of Linstowia (Cestoda: Anoplocephalidae) from marsupials in Bolivia.

A new species of cestode of the genus Linstowia (Cestoda: Anoplocephalidae) is described from marsupials of the genera Thylamys and Monodelphis. The new species (Linstowia schmidti) differs from Linstowia iheringi Zschokke, 1904, in having a much smaller strobila and reduced number of proglottids, and in the distribution of the eggs in gravid proglottids. In Bolivia, cestodes of the genus Linstowia appear to have a restricted geographic distribution, occurring in marsupials only in southeastern Bolivia near the western margin of the Chaco. This host-parasite association may represent an ecological-historical relict.


Tikusnema javaense n. gen., n. sp. (Nematoda: Acuarioidea) and other nematodes from Rattus argentiventer collected in west Java, Indonesia.

Nematodes collected from the ricefield rat, Rattus argentiventer (Rodentia: Muridae), in Pusakanagara and Sukamandi, West Java, Indonesia, are reported. Tikusnema javaense n. gen., n. sp. (Nematoda:Acuariidae:Seuratiinae) is described from the small intestine. This new genus is distinguished readily from other genera of the subfamily Seuratiinae in having 4 strongly protruded cuticular leaves in the posterior cephalic portion and in having a pair of prominent cuticular ornamentations posterior to deirids. Besides T. javaense, Eucoleus bacillatus, Strongyloides ratti, Nippostrongylus brasiliensis, Syphacia muris, and Physaloptera sp. were detected.


Two new coccidian parasites from the slate-colored grosbeak (Pitylus grossus) of South America.

In July and August 1990, fecal samples from 2 slate-colored grosbeaks were collected from the rain forest of Ecuador. Upon examination, 2 new species of coccidia were discovered. Oocysts of Isospora pityli n. sp. are spheroid or subspheroid, 20.1 x 18.8 (20-20.5 x 17-20) microns, with a shape index of 1.07 (1.0-1.18) but lacking a micropyle, oocyst residuum, and polar granules. Sporocysts are ovoid, 14.7 x 9.4 (12-17 x 8-11) microns, with small nipplelike Stieda bodies, no substieda bodies, and residua composed of an amorphous cluster of coarse, nonuniform granules. Sporozoites each possess a large refractile body at 1 end and appear to be enclosed in a thin membrane within the sporocyst along with the residuum. Oocysts of L. formarum n. sp. are spheroid or subspheroid, 24.6 x 23.5 (21-27 x 20-25) microns, with a shape index of 1.05 (1.0-1.09) but with no micropyle, oocyst residuum, or polar granules. Sporocysts are ovoid, 15.7 x 11.3 (14-17 x 10-13) microns, with small, nipplelike Stieda bodies and large triangular or conical-shaped substieda bodies with irregular lower edges. Sporozoites each possess an oblong refractile body at 1 end and appear packed together randomly and enclosed in a membrane along with a spheroid residuum composed of fine, uniform granules.


Onchocerciasis in Minnesota cattle.

Umbilical hides from 536 dairy cattle in Minnesota were tested for the microfilarial stage of Onchocerca species to determine the distribution of onchocerciasis in the state. The infection was widespread as microfilariae were obtained from 214 (40%) of the animals, representing nearly all areas of the state. Adult Onchocerca parasites were collected primarily from nodules associated with tibial bones but also were found to a lesser extent within the gastrosplenic ligament. Specific identity of these organisms is unclear as they exhibit certain morphological features previously described as being characteristic of either Onchocerca gutturosa, Onchocerca lienalis, or Onchocerca stilesi.


A nucleic acid-based test for detection of Fasciola hepatica.

The use of nucleic acid techniques in the diagnosis of parasitic infection has become increasingly widespread. An oligonucleotide probe derived from a rRNA sequence was developed for the detection of Fasciola hepatica in its intermediate snail host Pseudosuccinea columella. Total RNA obtained from whole adult liver flukes was used in a polymerase chain reaction to isolate and amplify a region of approximately 650 base pairs in the small subunit rRNA. This portion of the ribosomal cDNA, which contains highly conserved regions as well as variable regions, was subcloned and sequenced. In comparison to known small subunit rRNA sequences, a sequence unique to F. hepatica was identified and an oligonucleotide probe (CS4) for detection of F. hepatica was developed. A northern blot analysis using CS4 successfully identified small subunit rRNA from F. hepatica. Slot-blot analysis determined that RNA derived from 5 miracidia can be detected with CS4. Moreover, a slot blot utilizing CS4 distinguished RNA derived from snails infected with F. hepatica from RNA of uninfected snails.


Minimizing ELISA background in the diagnosis of swine trichinellosis.

After confirming that long-term serum storage (frozen at -20 C for greater than 3 mo) causes optical density to drift upward, several modifications of an enzyme-linked immunosorbent assay (ELISA) protocol were evaluated to identify a protocol that would reduce background in porcine sera tested for trichinellosis. Modifications evaluated included blocking the antigen-coated ELISA plate with sample diluent containing 10% bovine serum albumin (BSA) or 10% nonfat milk powder (bovine lacto transfer optimizer or BLOTTO), diluting sera in sample diluent containing 10% BSA or 10% BLOTTO, and preincubating samples in sample diluent containing 10% BSA or 10% BLOTTO. Overnight preincubation (approximately 12 hr at 2 C) of fresh sera diluted (1:10) in sample diluent containing 10% BLOTTO significantly reduced background and improved the detection of experimentally infected pigs by enhancing positive-negative discrimination. When testing stored sera, the modified protocol effectively reduced the effect of storage and the kit revealed specificity of 98.4%; there was no loss in sensitivity. The effect of long-term storage at -20 C must therefore be considered when testing swine sera for trichinellosis by ELISA and possibly also when conducting other immunoglobulin assays. The modification described here may prove useful if there is no alternative to using serum stored for greater than 3 mo at -20 C.


Host-parasite relationships of longnose dace, Rhinichthys cataractae, from the Ford River, Michigan.

A total of 1,115 longnose dace, Rhinichthys cataractae (family Cyprinidae), were examined for parasites from May 1983 through October 1986 from 3 localities in the Ford River in Michigan's Upper Peninsula. Thirteen parasite species (1 Monogenea, 2 Digenea, 2 Cestoda, 4 Nematoda, 1 Acanthocephala, 3 Protozoa) infected dace. The parasite faunas of dace, taxonomically and in species number, were similar between localities. Posthodiplostomum minimum minimum, Neascus sp., and Rhabdochona canadensis were the most common helminths infecting dace from each locality. The first 2 species did not exhibit consistent seasonal infection patterns between years, whereas the prevalence and mean intensity of R. canadensis in dace from the downriver locality were higher in summer 1983, 1984, and 1985. The intensity of infection of each of these helminth species significantly increased with host length. The prevalences and mean intensities of P. m. minimum, Neascus sp., and R. canadensis as well as the helminth infracommunity diversity were highest in dace from the upriver locality. The major factors that influenced parasite intensity were environmental factors that occurred when and where a fish began its life, the sequence of events that occurred in each habitat the fish encountered during its life, and the length of exposure (age of fish). Dace have isolationist helminth infracommunities arising from factors including ectothermy, a simple enteric system, restricted vagility, and being gape-limited. Allogenic helminths with indirect life cycles predominate in the depauperate helminth fauna of dace.


Reduced splash dispersal of bovine parasitic nematodes from cow pats by the dung beetle Diastellopalpus quinquedens.

From a thoroughly mixed portion of cattle feces with Cooperia sp. eggs, 1-kg artificial pats were placed in 6 buckets containing 6 kg of soil each. Ten dung beetles, Diastellopalpus quinquedens, were added to each of 3 buckets. The remaining 3 buckets served as controls without beetles. When infective parasite larvae (L3) had developed in the cow pats indoors, the following procedure was followed. During occasions of rainfall each bucket was placed outdoors in the center of a wider and higher container. When the rain stopped all buckets were brought indoors, and infective larvae spread by splash droplets during the rain were collected in the containers and counted. After 33 days, the remaining dung on the soil surface in buckets with dung beetles constituted only 38% of that in the controls. Moreover, the number of L3 in feces left on the soil surface in the buckets with beetles was reduced by 88%, presumably due to beetle activity. This may explain the 70-90% reduction in splash dispersal of L3 of Cooperia sp. from cow pats attacked by beetles. The dung-burying activity of the beetles did not result in increased numbers of L3 in the soil under the cow pats, suggesting that many of the parasites in buried feces were destroyed.


Chemotactic response of macrophages to Acanthamoeba castellanii antigen and antibody-dependent macrophage-mediated killing of the parasite.

The chemotactic potential of antigens of Acanthamoeba castellanii for macrophages and the ability of naive and immune rat peritoneal macrophages to kill A. castellanii in vitro were assessed. The amoebolytic capacity of immune rat serum and complement was also examined. No parasite was killed in the presence of heat-inactivated naive rat serum. Low numbers of parasites were lysed in the presence of heat-inactivated immune rat serum, whereas significantly greater numbers of parasites were lysed in the presence of nonheat-inactivated naive and immune rat serum. Macrophages from naive rats were capable of lysing some parasites. However, the amoebolytic capability of these cells was significantly increased in the presence of serum from immune rats. Regardless of the source of serum used, macrophages from immune rats demonstrated about twice the amoebolytic proficiency of cells from naive rats. Macrophages from naive rats showed their highest capacity for lysing amoebae when incubated in the presence of gamma interferon and immune rat serum. The greatest overall proficiency in lysing parasites was displayed by cells from immune rats incubated with A. castellanii in the presence of gamma interferon and nonheat-inactivated serum from immune rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Incorporation of [3H]hypoxanthine by short-term cultured Theileria sergenti and its inhibition by drugs.

Red blood cells parasitized with Theileria sergenti were cultured in vitro for a short period in microplate wells. Addition of [3H]hypoxanthine to cultures enabled us to titrate the intraerythrocytic parasite growth because a significant amount of [3H]hypoxanthine was incorporated into the parasitized red blood cells (PRBC) in proportion to the number of PRBC. The incorporation of [3H]hypoxanthine was almost completed in an early phase of incubation. Bovine peripheral blood leukocytes incubated with lysate of PRBC did not incorporate [3H]hypoxanthine, indicating that contaminating leukocytes were not involved in the incorporation in the PRBC culture. The incorporation of [3H]hypoxanthine was decreased markedly by the addition of certain anti-parasitic drugs such as chloroquine, quinine, and pyrimethamine. This inhibition test was performed quantitatively with high sensitivity. Based on these results, this short-term culture seemed to be useful for drug screening or studying the mechanisms of theilerial infection. However, anti-T. sergenti antibodies failed to inhibit the [3H]hypoxanthine incorporation.


Developmental biology and migration of Strongyloides ratti in the rat.

Most recent authors suggest that larvae of the threadworm, Strongyloides ratti, migrate from a cutaneous infection site to the small intestine via the naso-frontal region of the head. However, the proportion of larvae that successfully reach the intestine having followed this pathway had not been determined. Using compartmental analysis we have obtained a comprehensive quantitative description of larval migration during a primary infection of rats with S. ratti. Mean residence times in organs through which larvae migrate were calculated and the proportion of the larvae arriving in the small intestine via the head was estimated as 0.5-0.86, depending on the mathematical model used to generate the estimate. With 50% or more of successful larvae using this pathway it is now reasonable to recognize the "head route" as an important migratory pathway for larvae traveling to their intestinal predilection site. During the first 12 hr in the host, larvae were difficult to recover from the skin and subjacent muscles of the infection site. Apparently, upon invading a host, many larvae enter a quiescent phase during which Baermannization, an active recovery technique, fails to recover them. Infections were short-lived, with the daily finite mortality rate for adult worms attaining 50-80% on day 16 of infection.


Effect of cobalt-60 irradiation on the infectivity of Paragonimus westermani metacercariae.

A study was made to observe the effect of cobalt-60 irradiation on the viability of Paragonimus westermani metacercariae in Sinopotamon chekiangense crabs. The crabs were collected in mountain regions of the Zhejiang Province of China in which paragonimiasis is endemic. Adult cats and albino mice were infected with metacercariae irradiated at different doses. Dissection of the host animals was conducted 90 or 30 days, respectively, after infection for recovery of lung flukes. Anti-metacercariae antibody in infected mice was measured by enzyme-linked immunosorbent assay (ELISA). Results showed that metacercariae were unable to grow into adult worms in cats after exposure to gamma irradiation at a dose of 0.10 kGray. However, a small number of metacercariae exposed to a dose of 2.0 kGray excysted and survived in 1 mouse for 30 days. No worm was recovered from mice when the metacercariae were irradiated at a dose of 2.5 kGray. Seropositive results by ELISA were obtained when the mice were infected with metacercariae irradiated at doses ranging from 2.0 to 3.5 kGray.


Characterization of the intracellular melanization response in Anopheles quadrimaculatus against subperiodic Brugia malayi larvae.

Intracellular melanization, a defense or an immune response in the thoracic muscle cells, was investigated in a refractory strain of Anopheles quadrimaculatus infected with larvae of Brugia malayi. In mosquitoes fed on B. malayi-infected jirds, intracellular melanization against first-stage larvae (L1) was better expressed when fewer than 40 microfilariae reached the thoracic muscle cells than when more than 40 microfilariae reached the thoracic muscle cells. This result suggests that when large numbers of microfilariae invade the thoracic muscle cells, the immune response of the mosquito may become overloaded. Intracellular melanization response against L1 in the thoracic muscle cells also showed a significant decrease in older females (14-16-day-old) as compared to the younger ones (4-9-day-old). A comparison is made between intracellular and extracellular responses of mosquitoes to filarial larvae. It is significant that in both cases high rate of infection can reduce both the number and percentage of larvae melanized.


Effects of eimerian (Apicomplexa: Eimeriidae) infections on nutrient assimilation in the Wyoming ground squirrel.

Effects of infection with mixed species of Eimeria (E. callospermophili, E. morainensis, and E. beecheyi) on the digestive physiology of Wyoming ground squirrels (Spermophilus elegans) are described. Infected and uninfected squirrels were administered arginine and methionine in saturated glucose solution. Blood was drawn at time 0 and 30 min postadministration. Significant differences were not found between infected and uninfected squirrels in plasma glucose, arginine, or methionine assimilation. In a second experiment, infected and uninfected squirrels were fed a food slurry of known caloric value. All feces were collected for 24 hr postfeeding. Differences were not detected in fecal caloric content or digestive efficiency. These results in conjunction with results reported in the literature suggest a reassessment of the "parasitic" nature of these squirrel symbionts. We propose that associations of some Eimeria species and hosts that evolve under natural conditions are examples of parasite-host interactions that often evolve toward commensalism.


Identification and partial purification of a lectin on the surface of the sporozoite of Cryptosporidium parvum.

A human-derived isolate of Cryptosporidium parvum from a symptomatic patient with the acquired immunodeficiency syndrome was expanded in vivo by infecting a neonatal calf with 10(8) oocysts. Sporozoites were isolated from 4 x 10(10) oocysts harvested from this single infection, and the characteristics of mixed hemagglutination (HA) with rabbit erythrocytes were determined. Sporozoite HA was inhibited by bovine submaxillary mucin (BSM), hog gastric mucin, and orosomucoid, but not by simple sugars, including sialic acid. Carbohydrate-inhibitable HA (lectin) activity increased with sporozoite lysis and was associated with the sporozoite membrane fractions. The ability of intact sporozoites to form rosettes around erythrocytes indicates that the HA (lectin) is, at least in part, present on the parasite surface. Hemagglutination (lectin) activity was partially purified from sporozoite lysates by affinity chromatography with BSM coupled to Sepharose-4B. Best elution was obtained with ethylene glycol and NaCl, which resulted in enrichment of 6 bands compared to the crude starting lysate (Mr = 60, 24, 22, 20, and 15 kDa and a 40-kDa doublet). Our results indicate that an HA (lectin) activity is present on the surface of intact sporozoites where it could play a role in cell-to-cell interactions with eukaryotic targets.


Response to ivermectin treatment of parasitic stages of Haemonchus contortus resistant or susceptible to ivermectin.

Two groups of 33 helminth-naive lambs were infected with 5,000 L3 of an ivermectin-resistant or -susceptible strain of Haemonchus contortus (groups R and S). On days 6, 10, 16, and 21 postinfection, 5 animals from each group were chosen at random and orally treated with 0.2 mg/kg of ivermectin. On each occasion, 2 randomly selected lambs from each group were also killed to determine the number and stage of development of the worms present at the time of treatment. These necropsies revealed that by day 6 early and late fourth-stage larvae were present, whereas on day 10 the early fifth stage had been reached; by days 16 and 21 all worms had reached the adult stage. Necropsies on day 28 postinfection revealed that although animals treated at day 6 had 26.3% fewer worms than the controls, there was no significant difference (P greater than 0.05) between worm burdens from any of the animals infected with the R strain and treated at different times after infection when compared with the untreated controls. With ivermectin significant reductions were obtained in the worm burdens of the animals infected with the susceptible strain; these were reduced by 96% when treatment was given on day 6 against fourth-stage larvae and 98.9% when the drug was given on day 21 against adult stages. From these results it is clear that resistance to ivermectin in this strain of H. contortus is present as early as the fourth larval stage.


Metazoan parasites of the mountain beaver, Aplodontia rufa (Rodentia: Aplodontidae), from Washington and Oregon, with a checklist of parasites.

Four species of fleas, 2 species of mites, and the cysticerci of Taenia tenuicollis were collected from mountain beavers, Aplodontia rufa, collected in Washington and Oregon. A summary of the metazoan parasites reported from A. rufa is included.