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Mitochondrial DNA isolated from white blood cells was investigated in families suffering from Leber's hereditary optic neuropathy. A recently described mutation at nucleotide position 11778 was present in 5 out of 12 families and heteroplasmic mitochondrial DNA was observed in 2 of these 5 families. A rapid shift in genotype was found in one of the families with heteroplasmy: the grandmother had 60 percent mitochondrial DNA mutated at nucleotide position 11778, the mother 55 percent, and the two sons at least 95 percent. These data indicate that the number of mitochondrial DNA molecules transmitted to the progeny passes a developmental bottleneck, as previously proposed to occur in bovine oogenesis.

This paper describes the analysis, by a highly sensitive and specific enzyme immunoassay (EIA), of AcSDKP, a tetrapeptide recently isolated from fetal calf bone marrow and subsequently purified and identified which substantially inhibits entry into cycle of hematopoietic pluripotent stem cells (CFU-S). This molecule has a marked protective effect in mice during anticancer chemotherapy with phase-specific drugs and plays an essential role in maintaining CFU-S out of cycle in normal mice. Using acetylcholinesterase-AcSDKP conjugate as tracer, rabbit specific antiserum and 96-well microtiter plates coated with a mouse monoclonal anti-rabbit IgG antibody, this EIA allows detection of AcSDKP at 15 fmol levels with a coefficient of variation less than 10% in the 50-500 fmol range. When combined with high-performance liquid chromatography, this assay clearly reveals the presence of this peptide in normal human white blood cells whereas in supernatant from cultured lymphocytes and in plasma the immunoreactive material is distinct from standard AcSDKP.

To examine the role of DNA methylation in breakpoint location of chromosomal translocation, HpaII sites in and flanking the M-bcr on chromosome 22 were mapped in DNA from blood granulocytes and lymphocytes, bone marrow cells, thymic tissue, and spermatozoa from normal individuals. Allelic HpaII sites were identified clustered in a 600-base pair genomic area of the M-bcr. Bone marrow cells and blood granulocyte DNA showed identical allelic patterns. Thymic tissue and blood lymphocytes showed identical allelic patterns distinct from bone marrow cells and blood granulocytes. Spermatozoa showed a third methylation pattern. In all individuals, the HpaII sites were present within the BamHI/BglII fragment of the M-bcr, the same area associated with high breakpoint frequency in chronic myelogenous leukemia (CML). Three of 15 patients with chronic phase CML showed fully methylated rearranged BglII/BglII M-bcr restriction fragments not seen in normal bone marrow cells. These methylation patterns of the M-bcr may be important in CML breakpoint location and may be a marker for tissue differentiation.

Taurine (T) was reported to be at high concentrations in human leukocytes. It was proposed that T is a scavenger for chlorinated oxidants produced by the myeloperoxidase system of monocytes and neutrophils. Hypotaurine (HT) would be a more effective scavenger, and HT could also detoxify products of bromide or iodide oxidation produced by the eosinophil peroxidase system. Methods previously used to measure T in leukocytes might oxidize HT to T or fail to separate T and HT. Therefore, we examined T and HT content, uptake, and biosynthesis in isolated blood cells and cultured tumor cells derived from hematopoietic/lymphoid cells. Platelets and all leukocytes including monocytes, lymphocytes, neutrophils, and eosinophils had high T levels (10-20 mM), and all except eosinophils had substantial HT levels (0.3-1 mM). Intracellular levels were 500-times higher than in plasma. Erythrocytes were the only blood cells with low levels of both T and HT. Tumor cells from lymphoid (CCRF-CEM) and myeloid (HL-60, K-562, RWLeu4, HEL) lineages took up and concentrated T and HT from the bovine calf serum in the culture medium, and intracellular levels were similar to those in leukocytes. When cells were cultured in HT-supplemented media, HT almost completely replaced T, and HT was not converted to T. Levels of T were not raised by culturing cells with possible precursors, but HT levels were raised when cysteine sulfinic acid was present. Washed tumor cells took up T and HT by way of a beta-amino acid transport system, but uptake by leukocytes was very low. Therefore, leukocytes may acquire T and HT by active uptake rather than biosynthesis, and uptake may be completed during differentiation in the bone marrow. Though HT is low relative to T, HT levels may be sufficient to protect leukocytes from toxic oxidants.

Lactotransferrin was highly purified from lysates of human neutrophilic leucocytes by immuno-affinity chromatography. A comparative analysis of the molar carbohydrate compositions of human leucocyte lactotransferrin and human milk lactotransferrin reveals that the glycans of leucocyte lactotransferrin differ essentially by the absence of fucose residues. Structural analysis combining methylation-mass spectrometry and 400 MHz 1H-n.m.r. spectrometry of oligosaccharide alditols released from human leucocyte lactotransferrin shows the presence of two disialylated and non-fucosylated biantennary glycans of the N-acetyl-lactosaminic type. These results question a previously proposed mechanism for hyposideraemia in which the leucocyte lactotransferrin was involved and in which the fucose residues played a key role.

We developed a competition binding assay for estimation of the intracellular inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) and optimalized it for the measurement of the Ins(1,4,5)P3 content of human blood leucocytes. The present method is considerably cheaper and requires five times fewer cells than the commercial Ins(1,4,5)P3 kit. The mean Ins(1,4,5)P3 content of human blood monocytes, granulocytes, and lymphocytes amounted to 3.3 +/- 1.2 microM, 3.1 +/- 1.4 microM, and 4.6 +/- 1.5 microM, respectively. After stimulation with formyl-methionyl-leucyl-phenylalanine (f-MLP) the Ins(1,4,5)P3 content of human granulocytes and monocytes increased 2-3 times within 10 sec and then gradually decreased, returning to basal values at 60 sec. Lymphocytes did not respond to f-MLP with an increase in their Ins(1,4,5)P3 content.

Prolidase (peptidase D) catalyzes hydrolysis of the di- and tripeptide with carboxyl-terminal proline and plays an important role in recycling proline in various cells and tissues. By using human prolidase cDNA as a probe, a chromosomal gene related to prolidase was isolated from human gene libraries. The human prolidase gene is over 130 kilobases long and is split into 15 exons. All of the splice donor and acceptor sites conform to the GT/AG rule. The transcription initiation site was determined by nuclease S1 mapping and primer extension and was located 131 bases upstream from the initiation codon. A "CAAT" box-like sequence was present 67 bases upstream from the cap site, but there was no "TATA" box-like sequence. There were seven sets of sequences resembling the transcription factor Sp1 binding sites. Four were upstream from the cap site, and three were downstream. We also analyzed findings in patients with prolidase deficiency with respect to major gene re-arrangement. Several hundred base deletions, including the 14th exon, were identified. Knowledge of the gene structure of human prolidase will facilitate further studies on the expression and regulation of this gene and provide necessary information for analyses of mutations in patients with this deficiency.

To date, analysis of the DNA content of head and neck squamous cell carcinomas has relied on the homogenation of the entire tissue specimen and subsequent staining and quantitation of the naked nuclei. This methodology does not make allowance for the extremely variable nature of these tumors with respect to their cellular composition. Further, by destroying the cytoplasm and cell membranes, this methodology makes it impossible to distinguish the DNA content of the tumor cells from that of the background stromal and inflammatory cells. The authors present a methodology for the selective exclusion of inflammatory cell infiltrates from the DNA analysis of these tumors. Using this technique, it has been found that exclusion of the inflammatory cells allows the investigator to look more specifically at the malignant cell population. This has been most helpful in those samples in which the tumor cells have been diploid or near-diploid. With this technical refinement, the relationship between DNA ploidy and clinical prognosis may be more accurately assessed.

The effect of environmental pollution on DNA adducts in humans was analysed in a highly industrialized area of Poland. Coded samples of white blood cell DNA were analysed by 32P-postlabelling and immunoassay from three populations: coke workers, exposed occupationally to high levels of polycyclic aromatic hydrocarbons (PAHs); residents of the towns around cokeries (local controls); and residents from rural Poland (countryside controls). Local controls exhibited adduct levels and patterns similar to those of coke workers, while the levels in rural controls were 2-3 times lower. The results, based on coded samples and two different assays, suggest that environmental pollution is likely to contribute to the adduct levels in local controls. Furthermore, the results show that the levels of aromatic adducts in white blood cell DNA do not linearily relate to ambient air levels of PAHs but other sources such as food may be important contributors.

Four groups of patients with long-term inhalation exposure to formaldehyde (HCHO) were compared with controls who had short-term periodic exposure to HCHO. The following were determined for all groups: total white cell, lymphocyte, and T cell counts; T helper/suppressor ratios; total Ta1+, IL2+, and B cell counts; antibodies to formaldehyde-human serum albumin (HCHO-HSA) conjugate and autoantibodies. When compared with the controls, the patients had significantly higher antibody titers to HCHO-HSA. In addition, significant increases in Ta1+, IL2+, and B cells and autoantibodies were observed. Immune activation, autoantibodies, and anti-HCHO-HSA antibodies are associated with long-term formaldehyde inhalation.

The presence and amount of the anti-inflammatory protein lipocortin 1 was determined in plasma and peripheral blood leucocytes by a highly specific, enzyme-linked immunosorbent assay. Within 120 min of a single intravenous dose of 100 mg hydrocortisone, the intracellular concentrations of lipocortin 1 in peripheral monocytes in 7 of 8 healthy men increased by a median of 225% (range 129-507%) compared with pretreatment levels, and mononuclear cell-surface lipocortin increased by a median of 224% (range 76-483%). Placebo injections had no effect. There was no increase at any time in free plasma or polymorph-associated lipocortin. In 3 of 4 subjects, induction of lipocortin was also observed when whole unseparated blood was incubated in vitro after steroid administration, but cells which had first been isolated and purified were refractory to such induction. Thus rapid changes in the concentration of an active anti-inflammatory protein can occur in man after normal therapeutic doses of hydrocortisone.

We have undertaken a study among coke-oven workers to test the feasibility of an enzyme-linked immunosorbent assay with anti-trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene- DNA antibodies for monitoring occupational exposure to polycyclic aromatic hydrocarbons (PAH). Coke-oven workers are occupationally exposed to relatively high levels of PAH and are at increased risk for lung cancer. Three blood samples were collected from each of the 56 coke-oven workers exposed to PAH and 44 unexposed workers employed in a steel-rolling factory of the same plant. In addition, PAH levels were measured in ambient air by personal sampling, and the excretion of 1-hydroxypyrene in urine was also measured on 3 consecutive working days. All participants were interviewed regarding working conditions, personal hygiene, and smoking habits. The results showed that the coke-oven workers were exposed to substantial concentrations of atmospheric PAH (1-186 micrograms/m3), including benzo[a]pyrene (0.1-7.8 micrograms/m3) and pyrene (0.6-23.6 micrograms/m3). Both benzo[a]pyrene and pyrene were shown to be representative for the whole group of PAH. Forty-seven percent of the coke-oven workers had detectable levels of PAH-DNA adducts in their white blood cells, compared with 30% of the controls. In both groups, smokers had significantly higher levels of PAH-DNA adducts than did nonsmokers. At one site, we found the correlation positive between DNA adducts and the duration of exposure (r = .47, P = .005). Generally, the correlation was not significant between PAH-DNA adducts in blood and the concentration of PAH in the air and 1-hydroxypyrene in urine.

Human interferon omega 1 (IFN-omega 1 = IFN-alpha II1) is a recently discovered protein structurally related to IFN-alpha and -beta; the biological activities of IFN-omega 1 and its physiological role are not known to date. We have purified IFN-omega 1 from preparations of human leukocyte IFN, derived from peripheral blood leukocytes induced with Sendai virus, by two sequential cycles of monoclonal antibody affinity chromatography. The resulting protein was at least 95% pure as determined by reverse-phase high performance liquid chromatography and showed an Mr of 24,500 upon sodium dodecyl sulfate-gel electrophoresis (theoretical Mr, 19,984). Amino acid sequence analysis revealed that only about 40% of the molecules have the NH2 terminus expected on the basis of the sequence similarity to IFN-alpha, whereas the others contain two additional amino acids. This difference probably results from variable cleavage of the pre-protein by the signal peptidase. No evidence for COOH-terminal heterogeneity was found. Essentially all IFN-omega 1 molecules are glycosylated; enzymatic deglycosylation resulted in a reduction of the Mr to 20,500. Experiments using several plant lectins indicated the presence of biantennary complex oligosaccharides containing neuraminic acid. Two major peaks were observed upon chromatofocusing, with isoelectric points of 8.1 and 8.5. The specific antiviral activity of purified IFN-omega 1 assayed on human cells was determined to be 2.7 x 10(8) IU/mg, similar to that of other human class I IFNs; potent antiviral activity was also observed on cells of bovine and ovine but not of equine or murine origin.

1. The zinc status and drug-metabolizing ability of 15 patients with histologically diagnosed hepatic cirrhosis were studied. Zinc status was assessed using both serum and leucocyte zinc concentrations, and drug-metabolizing ability was assessed by antipyrine kinetics. 2. Patients with cirrhosis were found to have lower serum and leucocyte zinc concentrations when compared with a healthy control group. 3. Leucocyte zinc content and antipyrine clearance were correlated. Those patients with the lowest leucocyte zinc content had the greatest impairment of drug metabolism. Antipyrine elimination and serum zinc concentrations were not correlated. 4. Leucocyte zinc concentrations and antipyrine clearance were not influenced by the severity of liver dysfunction, as assessed by using the Child Turcotte classification. 5. These results suggest that tissue zinc depletion in some patients with hepatic cirrhosis may explain in part the impaired capacity to metabolize drugs.

A 1.9-kb fragment containing an interleukin-8 (IL-8) coding region was amplified by the polymerase chain reaction (PCR) from the genomic DNA of human lung giant cell carcinoma LU65C cells that produce LUCT/IL-8 with N-terminal sequence of AVLPR. The coding region was found to consist of 4 exons and 3 introns as identical as that of the gene of MDNCF/IL-8 lacking N-terminal AVLPR. PCR using genomic DNAs from human polymorphonuclear leukocytes and mononuclear cells also provided the same 1.9-kb fragment as that from LU65C genomic DNA. Thus, it seems likely that human cells possess IL-8 genes with the homogeneous coding region so that they may first produce the same mature protein with N-terminal AVLPR (= LUCT) which was then truncated.

Blood samples were obtained from volunteers who were occupationally exposed to polycyclic aromatic hydrocarbons in a Finnish iron foundry and from referents not known to be occupationally exposed to this class of chemical carcinogens. Aromatic adducts were determined in the deoxyribonucleic acid of white blood cells from the exposed workers with the 32P-postlabeling and immunologic techniques. There was a correlation between the estimated exposure in a particular job and the adduct levels. Jobs of men with high adduct levels (greater than 1 adduct/10(7) nucleotides in the postlabeling assay) included sand preparation, molding, shake-out, and transport. The adduct levels were low in men in pattern making, melting, and fettling. This study suggests that 32P-postlabeling and immunoassay may be useful in monitoring human exposure to known and previously unidentified environmental genotoxic agents.

Bacteriological examination of urine is sufficient to assert the presence of a urinary tract infection (UTI). Specimens must be collected under strictly aseptic conditions. The midstream jet method is the best, but when it is not feasible bladder puncture, sterile pouches or catheterization may be used. The urine obtained must be correctly preserved and transported. In the practitioner's office, UTI can be detected by reagent strips or agar-coated plates. In the laboratory, direct examination is used to count leucocytes and characterize Gram-positive and Gram-negative bacteria. Bacteriuria at 10(5)/ml is significant of infection. The micro-organisms most frequently responsible for UTI are Enterobacteriaceae (mostly E. coli), Proteus mirabilis, Klebsiella spp. and staphylococci (Staph. aureus, Staph. saprophyticus, Staph. epidermidis). In most cases disc sensitivity tests are indispensable, but bacteriological examination of urine is not always needed in women with uncomplicated lower urinary tract infection.

Consuming substantial quantities of n-3 fatty acids reduces atherogenesis in experimental models of atherosclerosis. The mechanisms of this beneficial effect remain uncertain. Monocyte-derived tissue macrophages are associated with atherogenesis, and inhibition of monocyte inflammatory activity could, hypothetically, be helpful in preventing atherosclerosis. We observed that stimulated human monocyte and/or macrophage production of superoxide and the occurrence of monocyte chemiluminescence, two indices of monocyte inflammatory activity, were significantly reduced by the ingestion of 6 g n-3 fatty acids/d for 6 wk. This effect was associated with a reduction of stearic and arachidonic acids whereas eicosapentaenoic and docosahexaenoic acid concentrations rose significantly. These results indicate that modest dietary n-3 fatty acid supplementation can reduce stimulated human-monocyte free-radical production and may impair the capability of macrophages derived from monocytes to promote oxidation of low-density-lipoprotein cholesterol and associated cellular toxicity.

A child who was grossly malnourished and who showed increased excretion of porphyrin and porphyrin precursor had normal activity of erythrocyte porphobilinogen deaminase (EC 4.3.1.8) and leukocyte protoporphyrinogen oxidase (EC 1.3.3.4). Clinical symptoms, coincident with the excretion of rose-colored urine, were consistent with the diagnosis of an acute porphyria. The disease resolved spontaneously after the withdrawal of carbamazepine and sodium valproate and the commencement of parenteral nutrition with subsequent carbohydrate loading. In addition to normal concentrations of enzyme activities, the patient is unusual in presenting before puberty and in having no family history of porphyria.

A study has been made of granulated metrial gland (GMG) cells and adjacent layer 1 cytotrophoblast in the labyrinthine placenta of the mouse. In those cellular associations in which both cell types appeared healthy with the light microscope, examination with the electron microscope identified features which may form part of the early stages in the interaction which can occur between GMG cells and some layer 1 trophoblast. These included microvillous processes from each cell forming an interdigitating network, a polarization of mitochondria in a GMG cell with interdigitating microvilli, platelets, and some GMG cells which appeared to form a specialized cell junction with a small lymphocyte. The latter observation indicates that a tripartite functional relationship may exist between lymphocytes, GMG cells and layer 1 labyrinthine trophoblast. In a quantitative analysis of GMG cells in the maternal blood spaces of placentae no significant differences in the numbers of GMG cells present in these spaces or involved in an interaction with layer 1 labyrinthine trophoblast were detected between inbred and outbred pregnancies. These results suggest that histocompatibility antigens are not significant factors in determining the frequency of interactions between GMG cells and trophoblast.

Despite being essentially hepatotropic, hepatitis B virus has frequently been detected in peripheral blood leukocytes. In most cases, viral DNA has been detected by hybridisation, though, occasionally, evidence of viral RNA and virus-specific proteins has been reported. Though definitive evidence is lacking, it appears likely that the virus is able to replicate in at least a subfraction of these cells. The possible consequences with respect to the transmission and reactivation of virus infections are discussed.

Human alpha interferon produced in Sendai virus-stimulated leukocytes was purified by immunosorbent affinity chromatography and subjected to subtype analysis. All subtypes in leukocyte culture supernatant were confirmed in purified preparation. One of these subtypes was reactive in Western blotting with antibody specific to alpha II (omega). N-terminal amino acid sequence analysis of this particular subtype showed addition of two amino acids to the N-terminus of alpha II predicted through cDNA studies. The apparent cleavage site for leader sequence of alpha II was different from the site common to all other alpha subtypes. This is the first report on the isolation of natural alpha II and its unique N-terminal amino acid sequence.

Single-agent chemotherapy with cisplatin or carboplatin can induce remissions in approximately 30% of previously treated patients with advanced stage ovarian cancer. Previous studies have shown that the extent of platinum-DNA adduct formation measured in WBC DNA of ovarian cancer patients treated with cisplatin or carboplatin is directly associated with disease response (Reed et al., Proc. Natl. Acad. Sci. USA, 84: 5024-5028, 1987). It has been unclear whether adduct level in WBC DNA is independent of known prognostic variables in this disease, or whether adduct level parallels a known prognostic variable that can be more easily monitored. In a cohort of 24 ovarian cancer patients treated with single-agent cisplatin or carboplatin, we retrospectively assessed the relationship between disease response, platinum-DNA adducts in WBC DNA, and each of eight prognostic variables by both univariate analysis and multivariate analysis. The prognostic variables evaluated included: response to previous treatment, Karnofsky status, total platinum dose prior to current therapy, stage of disease, age, bulk of disease at initiation of therapy, histological type, and histological grade. By univariate analysis, adduct level was strongly associated with disease response (two-sided P = 0.0058), with the next strongest associations with disease response being held by Karnofsky status (P = 0.125), stage of disease (P = 0.189), response to previous treatment (P = 0.352), total previous platinum dose (P = 0.358), and age (P = 0.374). No significant associations were found between adduct level and histological type or histological grade. Further, when patients were stratified by the number of cycles studied (one cycle, two cycles, or three cycles), higher levels of adduct were consistently seen in those patients responding to therapy. We conclude that, in this small cohort of refractory ovarian cancer patients treated with single-agent cisplatin or carboplatin, adduct level in WBC DNA appears to be more closely related to disease response than other previously identified prognostic variables.

The clinical diagnosis of allergies needs appropriate tools for the measurement of histamine release from leukocyte suspension of affected patients. In the last few years the established fluorometric procedure has been challenged by alternatives like histamine radioimmunoassays (RIA). Although measurements of histamine with the latter methods make this procedure more accessible as a diagnostic tool, further simplifications are always welcomed. A newly developed immunoenzymoassay for histamine is described in the present work and compared to a recent radioimmunoassay. Sensitivity and specificity are shown to be comparable in both procedures.

The relationship between ascorbic acid status and the urinary excretion of hydroxyproline was examined in 11 healthy male subjects fed an ascorbic acid-deficient diet for 14 wk while in a metabolic unit. The diet provided 5 mg ascorbic acid/d and was supplemented with ascorbic acid to give intakes of 65 mg/d (2 wk), 5 mg/d (4 wk), 605 mg/d (3 wk), 5 mg/d (4 wk), and an average 375 mg/d (1 wk). The urinary excretion of hydroxyproline increased by an average of 16% and 30% after the first and second depletion periods, respectively, and decreased to baseline values after supplementation with normal or high doses of vitamin C. Significant (p less than 0.05) inverse correlations were found between urinary hydroxyproline and plasma, red cell, and leukocyte ascorbic acid. These results show that urinary hydroxyproline excretion increases during human vitamin C deficiency but that this effect is not strong enough to provide a reliable marker of mild vitamin C deficiency.

AIDS in children is usually caused by vertical transmission of human immunodeficiency virus type 1 (HIV-1). Aborted eight-week fetal and placental tissue from HIV-1 positive and negative (enzyme-linked immunosorbent assay and Western blot) women was analysed by immunocytochemistry and in-situ hybridisation. Maternal decidual leucocytes, villous trophoblastic derivatives, villous mesenchymal cells, and embryonic blood cell precursors in tissues from seropositive patients all stained for HIV-1 antigen and hybridised for nucleic acids. These observations suggest that a cytological pathway for vertical transmission of HIV-1 is established by eight weeks gestational age.

Three novel pteridines have been isolated from the urine of patients with a new variant of 6-(L-erythro-1',2'-dihydroxypropyl)-5,6,7,8-tetrahydropterin (tetrahydrobiopterin) deficiency, showing hyperphenylalaninemia. From the results of high performance liquid chromatography, oxidative degradation, and gas chromatography-electron impact mass spectrometry, their structures were identified as 7-(D-erythro-1',2',3'-trihydroxypropyl)-pterin (7-neopterin), 7-(L-erythro-1',2'-dihydroxypropyl)-pterin (7-biopterin), and 6-oxo-7-(L-erythro-1',2'-dihydroxypropyl)-pterin (6-oxo-7-biopterin). The ratio of biopterin to 7-biopterin in the patients' urines was 1:1, and after oral loading with tetrahydrobiopterin, 7-biopterin excretion rose parallel to biopterin. This finding suggests that 7-substituted pterins may be formed endogenously by a yet unknown isomerization reaction. The cause of hyperphenylalaninemia is still unclear. The activities of the enzymes involved in tetrahydrobiopterin biosynthesis and regeneration were found to be normal in the patients, and no effect of 7-biopterin on these enzymes was observed in vitro. However, compared with the normal cofactor, tetrahydrobiopterin, the Km values of tetrahydro-7-biopterin for phenylalanine hydroxylase and dihydropteridine reductase are 20 and 5 times higher, respectively.

The relationship between metabolic control and leukocyte glycogen content in diabetes mellitus was re-evaluated, blood glycogen being measured by an enzymatic procedure. In 30 healthy subjects, fasting blood glycogen averaged 50.6 +/- 2.8 mg l-1 or 7.45 +/- 0.42 ng 10(3)-cells-1, the latter value being unaffected during a 60-min period of induced hyperglycaemia. Comparable levels were found in 18 Type 1 insulin-treated diabetic patients (blood glycogen 50.4 +/- 4.6 mg l-1, leukocyte glycogen 6.92 +/- 0.50 ng 10(3)-cells-1), 6 insulin-treated diabetic patients presenting with chronic pancreatitis (blood glycogen 62.2 +/- 9.3 mg l-1, leukocyte glycogen 6.69 +/- 0.70 ng 10(3)-cells-1) and 12 Type 2 insulin-treated patients (blood glycogen 53.7 +/- 4.3 mg l-1, leukocyte glycogen 7.51 +/- 0.44 ng 10(3)-cells-1). In severely ketotic patients, leukocyte counts and blood glycogen (160.8 +/- 29.6 mg l-1, p less than 0.01 vs stable diabetic patients) were increased, but the leukocytic glycogen content was not significantly affected either before or during intensive insulin therapy and rehydration. The leukocyte glycogen content was abnormally low, however, in 9 untreated Type 2 diabetic patients (5.29 +/- 0.39 ng 10(3)-cells-1, p less than 0.02 vs healthy subjects) and abnormally high (10.77 +/- 0.65 ng 10(3)-cells-1, p less than 0.005 vs healthy individuals) in 30 Type 2 patients treated by sulphonylurea, alone or in combination with insulin. No correlation was found between leukocyte glycogen and either fasting plasma glucose or HbA1c.(ABSTRACT TRUNCATED AT 250 WORDS)

P-glycoprotein, the molecular marker of multidrug resistance, was investigated by means of the specific monoclonal antibody C219 in circulating lymphocytes obtained from a patient affected by B-CLL, who had undergone chemotherapy with chlorambucil and CVP. Dot-immunobinding and immunoblotting revealed the presence of P-glycoprotein in the cell lysate, but the immunoperoxidase method and indirect immunofluorescence carried out with a fluorescence microscope and a cytofluorimeter failed to detect the protein. Some methodologic problems are discussed.

We measured the manganese content of whole blood, plasma, platelets, mononucleated cells, polymorphonucleated cells, and erythrocytes. Platelets and blood cells were separated from whole blood by use of discontinuous gradients of colloidal polyvinylpyrrolidone-coated silica (Percoll), and their manganese content was measured by Zeeman graphite furnace atomic absorption spectrophotometry, after digestion with nitric acid and hydrogen peroxide. Erythrocytes account for about 66% of the manganese in whole blood, whereas the "buffy coat"--platelets and leukocytes--accounts for about 30%. The "buffy coat" components turn over more rapidly than do erythrocytes, so their manganese content may better indicate the body's manganese status.

Analytic reversed-phase high performance liquid chromatography (RP-HPLC) was performed to separate from the crude lysed human leukocyte ultrafiltrate (LLU) its partially purified most immunoactive subfraction P2/II in vivo. Under conditions used, the highest degree of segregation of both. LLU and P2/II could be observed in the first, as well as in the last two fifth of the water-methanol gradient. The comparison of the RP-HPLC traces of LLU and P2/II suggests that probably some hydrophilic components of LLU have been removed or--at least--diminished. The preliminary amino acid analysis (AA) of the selected peaks showed that none of them lacks Gly, Ser, and Glu. Of the basic amino acid residues Lys has been found with relatively many peaks while hydrophobic as well as aromatic amino acids have been represented very modestly. Further study is warranted in order to determine better the bearings of presented findings for the in vivo situation.

Blood pressure and leucocyte sodium content were measured in black Jamaican patients with pre-eclampsia and pregnant controls. Similar measurements were made in a group of mothers who had pre-eclampsia 36-38 weeks previously and their controls. An increase in cell sodium accompanied the high blood pressure in patients with pre-eclampsia. There was also some elevation of both blood pressure and cell sodium in patients who had had pre-eclampsia 36-38 weeks previously. These findings suggest that disturbances of cellular electrolytes continue into the post-partum period. Changes in intracellular sodium may be important in the pathogenesis of pre-eclampsia as they parallel the increase in blood pressure levels.

Placental anticoagulant protein-I (PAP-I), a member of the lipocortin protein family, is a potent in vitro anticoagulant whose in vivo function is unknown. Very low levels of PAP-I were present in plasma of normal volunteers (0 to 5 ng/ml) and in randomly chosen plasma specimens from hospitalized patients (0 to 28 ng/ml). Review of selected hospital records did not reveal any single clinical entity that correlated with plasma levels. PAP-I was also found in amniotic fluid (12 to 107 ng/ml) and in conditioned medium of cultured endothelial cells (49 +/- 20 ng/ml). Gel filtration experiments showed that PAP-I was intact and uncomplexed in plasma and amniotic fluid. The protein was fairly abundant intracellularly: 4080 +/- 2560 ng/mg total protein in cultured umbilical vein endothelial cells; 178 +/- 109 ng/mg in platelets; 564 +/- 384 ng/mg in leukocytes; and 8.4 +/- 4.3 ng/mg in erythrocytes. The levels of PAP-I increased in platelet-rich plasma after stimulation of platelets with arachidonic acid but not after stimulation with ADP, epinephrine, thrombin, ristocetin, or collagen. These data suggest that PAP-I probably does not function as a circulating natural anticoagulant in normal persons.

Insulin participates in regulating ovarian function in normal and in pathological states. This effect of insulin may be mediated by ovarian insulin receptors. We have previously characterized human ovarian insulin receptors and began to examine their regulation in vitro. The present study examines regulation of human ovarian insulin receptors in vivo. Stromal ovarian tissue was obtained from 21 women during an indicated surgical procedure. Ten women were premenopausal and 11 were postmenopausal. Specific 125I-insulin binding to stromal ovarian fragments ranged from 2.5% to 7.3%/mg protein. 125I-insulin binding to stromal fragments correlated positively with 125I-insulin binding to circulating leucocytes (r = .57; P less than .01). When postmenopausal and premenopausal women were analyzed separately, this relationship persisted in postmenopausal women (r = .70; P less than .05), but not premenopausal women. 125I-insulin binding to stromal ovarian fragments correlated negatively with age (r = -.63; P = .005). 125I-insulin binding to stromal ovarian fragments tended to correlate negatively with plasma insulin levels in postmenopausal women (r = -.67; P = .06), but not in premenopausal women. Plasma insulin levels correlated negatively with serum SHBG (r = -.62; P = .003). The percent free testosterone levels correlated positively with plasma insulin levels in premenopausal women (r = .95; P = .0001), but not in postmenopausal women.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular diagnosis of disorders of follicle-stimulating hormone (FSH) production may become possible now that the gene for FSH beta has been characterized. Restriction-fragment length polymorphism (RFLP) analysis provides a means of organized search for molecular variants of FSH. The purpose of this study was to screen controls for the presence of RFLPs using the deoxyribonucleic acid (DNA) probe pFSH beta -1.4. Genomic DNA was digested with 12 different restriction endonucleases; Southern blots were constructed and hybridized to pFSH beta -1.4. No polymorphisms were identified with 11 enzymes. Three of 24 (12.6%) Hind III digests demonstrated a polymorphic fragment of either 5.2, 4.7, or 4.3 kb. These are the first RFLPs identified for the FSH beta gene with pFSH beta -1.4. RFLPs for FSH beta constitute the first step in the molecular analysis of disorders of FSH production.

Eighteen pediatric patients with cystinosis were treated with cysteamine. Treatment was started at a variable age, between 10 months and 7 years, and was continued for 6 months to eight years. Results were evaluated on renal function changes and growth. Whereas 65% of patients with cystinosis develop terminal renal failure before the age of ten years, three (16%) of our 18 patients given cysteamine started dialysis before the age of ten and all three had first received cysteamine only after 4 1/2 years of age. The plasma creatinine level was also lower in treated patients as compared with a control multicenter group. Growth was also significantly improved by treatment, especially in those children treated before the age of 26 months who were taller by 2 SD at age five and 2.5 SD at age eight than untreated controls. Some children who complied strictly with the treatment regimen had an normal stature (- 1 SD) at ten years of age. In conclusion, the effectiveness of cysteamine seems obvious in this group of patients if rigorous compliance with the drug dosing schedule is achieved.

Efficient transfection of eukaryotic cells is dependent on the purity of the transfected genomic DNA. In an attempt to obtain a more reliable method of DNA purification we have modified the widely used protocol of Blin and Stafford to include a treatment with guanidinium thiocyanate. The DNA obtained following the present protocol transfects eukaryotic cells with higher efficiency.

Cerebral MRI and CT findings were compared with various cerebrospinal fluid (CSF) parameters and clinical features in 75 patients with multiple sclerosis (MS). There were positive correlations between CSF albumin as a parameter of blood-brain barrier function and morphological parameters, namely the number of nonperiventricular foci and periventricular involvement demonstrated by MRI and ventricular enlargement as shown by CT. Apart from positive correlations between the number of nonperiventricular foci and CSF levels of IgA and IgG, including the IgG synthetic rate, no other correlations were found between CSF parameters (leucocyte count and immunoglobulin concentrations) and morphological findings. The unremitting-progressive type of MS was distinguished from the relapsing-remitting form by a lower CSF leucocyte count and a higher degree of disability, but there was no difference between the morphological findings in the two forms. Positive correlations were found between degree of disability and both periventricular involvement in the MRI and CT evidence of ventricular enlargement. In terms of correlations with the duration of disease, cerebral MRI proved to be far superior to CSF evaluations.

CD36 recognizes a 88 Kd glycoprotein, found on different cell populations involved in immunoregulation and are induced on keratinocytes by in vitro treatment with gamma-interferon. Therefore, we obtained skin biopsies from 48 patients with various dermatological diseases and from 5 healthy volunteers and stained these with monoclonal antibodies OKM5 (CD36), anti-HLA-DR and OKT6 (CD1a) using a three stage immunoperoxidase method. In normal skin, CD36 expression was not observed. In contrast, keratinocytes in diseased skin were CD36+. In most cases, the staining was restricted to the stratum granulosum and the stratum spinosum, but in psoriasis, squamous cell carcinoma and lymphomatoid papulosis, more extensive staining of keratinocytes was seen. In addition, CD36+ epidermal leukocytes were found in allergic patch-test infiltrates and in mycosis fungoides. The findings of CD36 expression by epidermal cells within a broad spectrum of dermatological diseases indicate a role for these cells in the regulation of immune reactions in the skin.

Haematological response and ascorbic acid (AA) status were evaluated in 32 young adult females (age 20-34 years) 8 weeks after supplementation with two levels of AA (50 and 100 mg). Both levels of AA intake produced a similar response marked by significant elevation (p less than 0.01) in Hb concentration, Hct level, RBC count, serum and leucocyte ascorbate concentrations. Ten weeks after withdrawal of the supplements, values for all parameters evaluated decreased significantly (p less than 0.01) to presupplementation levels. Haemoglobin concentration and leucocyte ascorbate remained relatively higher (p less than 0.01) than initial values in subjects who received 100 mg AA. This study showed that AA supplementation improved erythropoietic activity and AA status of our female population. However, the higher dose of AA (100 mg) sustained Hb concentration and tissue ascorbate longer after withdrawal of supplementation.

It is possible accurately to distinguish lymphocytes from other leukocyte populations in peripheral blood using the combination of fluorescence associated with CD45/CD14 and forward and orthogonal light scatter. By identifying the cell population of interest based on immunofluorescence, a light scattering window can then be drawn to include all (greater than or equal to 98%) of the lymphocytes. In this manner, maximal recovery of the lymphocytes within a sample can be consistently obtained. The combination of light scattering and immunofluorescence can also be used to define the purity of the gate. The identification of nonlymphocytes within the light scattering gate can then be used to establish an accurate denominator for the percent lymphocytes stained. Once the optimal data acquisition gate has been established and characterized, it is possible to correct subsequent analyses with that particular sample since the reactivity of monoclonal antibodies on monocytes and granulocytes can be accounted for once the nonlymphocytes have been identified as being within the acquisition gate.

Antisera raised against partially purified human leukocyte interferon in goat and horse were exhaustively adsorbed and purified. Affinity chromatography using these antibodies gave 10,000-fold purification in one step without losing interferon activity. Then enzyme immunoassay was established with these antibodies and utilized for subtype analysis. Crude and affinity purified leukocyte interferons showed identical profiles of antiviral activity and immunoreactivity when fractionated by means of reversed phase high performance liquid chromatography. These results suggested the possibility of providing mixture of subtypes of leukocyte interferon occurring in natural host response, with comparable purity of recombinant ones.

In seven patients undergoing elective cholecystectomy or vagotomy, helper and suppressor/cytotoxic T-cells were quantified by use of monoclonal antibodies (OKT) preoperatively and on postoperative days 2 and 7. The total number and percentage distribution of T-cells (OKT 3) did not change significantly in the study period and the total number of helper T-cells (OKT 4) was similar in the three tests, but the number of suppressor/cytotoxic T-cells (OKT 8) on postoperative days 2 and 7 showed significant rise from the preoperative value. Consequently, significant decrease in OKT 4/8 ratio was found 2 days (1.47 +/- 0.23) and 7 days (1.80 +/- 0.28) postoperatively, as compared with the preoperative ratio (2.85 +/- 0.44). Since reduced ratio of OKT 4/8 has been shown in disorders associated with diminished immunocompetence, our findings may be related to the increased susceptibility to bacterial infections seen in post-surgery patients.