Biomedical subjects
Publications and source records attributed to A Schmitz.
Flow cytogenetic is widely used since 1975, and essentially contributes to karyotype analysis and chromosome sorting. The principles of experimentation and its possibilities and limitations are now well known. Recently several new technologies have appeared. What attitude should the cytometrist adopt regarding PCR, microdissection of chromosomes, in situ hybridization, slit-scan flow cytometry or image analysis?
The principal end point in the evaluation of treatment in incipient and overt diabetic nephropathy is rate of decline in glomerular filtration rate (GFR). Therefore, information on reproducibility of GFR measurements is essential in the planning and evaluation of clinical trials. We studied reproducibility of GFR measurements in insulin-dependent and non-insulin-dependent diabetes mellitus patients using, respectively, a constant-infusion technique with urine collection and labeled iothalamate as a tracer marker and a single-shot procedure using Cr-EDTA, measuring the GFR from the decline in plasma level after bolus injection. The coefficient of variance in the insulin-dependent patients was from 7.5% to 8.8% with repeated measurements. In longitudinal studies with several measurements the mean coefficient of variances varied between 7.4% and 3.4%. In the non-insulin-dependent patients the coefficient of variances between two tests were 7.0% and 5.3% for normoalbuminuric and microalbuminuric patients, respectively. In cross-sectional studies as well as in longitudinal studies, it has been consistently shown that GFR is well preserved and at a supranormal level in patients with normoalbuminuria and microalbuminuria. A decline in GFR appears to start around the transition from microalbuminuria to overt diabetic renal disease, although more detailed studies are needed to support this finding. With regard to intervention trials, several studies document that microalbuminuria can be reduced by effective antihypertensive treatment, particularly with angiotensin-converting enzyme inhibitors, also in patients with normal or close to normal blood pressure. Preliminary results from long-term studies suggest that reduction in microalbuminuria in these patients is associated with preservation of GFR and, thus, apparently renoprotection. In patients with overt renal disease, it has been consistently shown that antihypertensive treatment reduces albuminuria as well as the rate of decline in GFR. This is also observed with combined treatment regimens, for instance beta blockers or angiotensin-converting enzyme inhibitors combined with diuretics, or the three types of drugs in combination.
We report the isolation, sequence, and identification of a cDNA encoding the human kinesin light-chain (KLC) protein. The cDNA molecule consisted of 276 nucleotides of 5' untranslated region, the complete coding sequence of 1,710 nucleotides, and 322 nucleotides of 3' untranslated region. It encoded a polypeptide of 569 amino acids and a deduced molecular mass of 64,789 daltons. The predicted secondary internal structure of the KLC molecule consisted of about 27 contiguous repeats, each of approximately 21 amino acid residues, and could be divided into three domains. The amino-terminal domain consisted of heptad repeats typical of the rod domain of several cytoskeletal proteins. The central and carboxy-terminal domains consist of 21-mer repeats. KLC mRNA was expressed in most tissues analyzed. The gene, which was expressed in bacteria and Chinese hamster ovary cells, was provisionally assigned to the long arm of human chromosome 14.
We report two cases of ischemic hepatitis in patients with alcoholic cirrhosis. In both, hepatic ischemia was induced by hemorrhagic shock and severe sepsis. Despite control of the bleeding and restoration of normal hemodynamics, liver failure deteriorated to hepatic coma and death in both cases. Ischemic hepatitis occurred in 1.5% of 130 consecutive cases of cirrhosis admitted for hemorrhage on our medical intensive care unit. Although cirrhotic patients run an increased risk of ischemic hepatitis, our experience and our review of the literature indicate that this condition is rare in these patients.
We present here a new PCR-based technique that allows the production of several micrograms of DNA from only 300 flow-sorted chromosomes. During the first two PCR cycles, the annealing temperature is decreased to 30 degrees C, and numerous random loci are amplified under nonspecific conditions. As demonstrated here for pig chromosomes 1 and 18, the PCR products may be used to identify the chromosomal content of the flow-karyotype peaks of any species by fluorescence in situ hybridization.
OBJECTIVE: To relate deterioration in kidney function to some potential cardiovascular risk factors in type II diabetic patients. RESEARCH DESIGN AND METHODS: Twenty-four normoalbuminuric and 13 microalbuminuric patients completed a 3.4-yr prospective observational study. Glomerular filtration rate, urinary albumin excretion rate, blood pressure, glycemic control, and lipids were measured on entry and at the end of the study. Of the patients, 19 normoalbuminuric and 8 microalbuminuric (73%) patients had no history of antihypertensive treatment. RESULTS: The glomerular filtration rate was significantly reduced during the follow-up period (-1.34 +/- 0.54 ml.min-1.1.73 m-2 [mean +/- SE], P < 0.02). The rate of decline varied considerably in normoalbuminuric and microalbuminuric patients (from -13.5 to 4.3 and from -7.0 to 4.2 ml.min-1.1.73 m-2 per year, respectively) but was on average not accelerated in normoalbuminuric or microalbuminuric patients (-1.3 +/- 0.7 and -1.5 +/- 0.8 ml.min-1.1.73 m-2 per year, respectively). Significant correlations were observed between the glomerular filtration rate fall rate and initial systolic blood pressure (r = -0.47, P < 0.01; patients without antihypertensive treatment: r = -0.42, P = 0.03) but not diastolic blood pressure. In a stepwise multiple linear regression analysis, baseline systolic blood pressure significantly determined the fall rate of the glomerular filtration rate (regression coefficient = -0.050, SE = 0.018, P = 0.011; patients without antihypertensive treatment: regression coefficient = -0.047, SE = 0.021, P = 0.030). CONCLUSIONS: In these type II diabetic patients neither normoalbuminuria nor microalbuminuria are at an average associated with an accelerated decline in kidney function. Still, systolic blood pressure is a determining factor for the rate of decline in the glomerular filtration rate. A longer follow-up time with consecutive glomerular filtration rate measurements are needed to determine the long-term implications of normoalbuminuria and microalbuminuria on kidney function in type II diabetic patients.
The merits of Thallium 201 radionuclide scanning of the thyroid mentioned as soon as 1976 by PALERMO have been confirmed in the 16 last studies published in the international literature. Over 1601 examinations, authors showed that any cold nodule (Tc 99 m or I 123) which preferentially fixes Thallium 201 as compared to surround thyroid tissue must be operated since some of them are thyroid cancer carriers, some others are carriers of a traditional follicular adenoma or Hurthle-cell adenoma, or a follicular adenoma associated with varied cell atypisms which make difficult the diagnosis between benign and malignant nodules. The analysis of the results published show a 91.3% sensitivity of this diagnosis test, all analysis methods being considered. In case of negative test, it allows eliminating the cancer risk by more than 97% (negative predictive value). Such an examination--as opposed to a cytology test on the isolated nodule only--is valid for any type of nodule, being isolated, multiple or a multinodular goitre where the degeneration risk is close to, or even higher than, that of isolated nodules (4, 22).
Phospholipase A2 (PLA2) was produced in E. coli by a recombinant technique using a synthetic gene coding for PLA2 found in synovial fluid (SF) of patients suffering from rheumatoid arthritis (RA). Polyclonal antibodies were produced against the recombinant synovial type PLA2 (syn-PLA2) in a rabbit. The IgG fraction of the antiserum was isolated and the specificity was tested by immunoblotting. The antiserum detected a protein with an apparent molecular weight of 15 kDa in extracts of cartilage, but did not react with PLA2 isolated from human pancreas or ascitic fluid. In immunohistochemistry, the anti-syn-PLA2 antibody decorated chondrocytes and matrix of articular, laryngeal and auricular cartilage, but did not decorate synovial tissue or its contained inflammatory cells in RA or other human tissues tested (pancreas, liver, thyroid, tonsil, lung, nasal polyp, skin, placenta, myometrium, bone). The results show that syn-PLA2 is present both in articular and extraarticular cartilage and support the view that PLA2 found in SF might originate from chondrocytes, and not from synovial lining or inflammatory cells.
The Conformity Analysis is a new procedure for objective recognition of specific defect patterns in automated perimetry. The "Conformity Index," the measure of conformity between the distribution of defect values and several standard defect patterns, is calculated from the ratio of global variance and mean variance in particular regions. Using 68 visual fields from 68 normal persons, we determined normal values and limits of the Conformity Index for seven standard defect patterns: hemifields right-left, hemifields upper-lower, quadrants, sectors, rings with 5 degrees and 10 degrees radius interval and the perimetric nerve fiber bundles of Weber and Ulrich. The evaluation of 148 visual fields (68 normal eyes, 80 eyes with chronic glaucoma and all stages of damage) by both the Conformity Index and two experienced clinicians revealed an identical result in 89%. Using the Conformity Analysis in 37 cases of pituitary adenoma, we could objectively identify quadrants, sectors and hemifields right-left to be the most common defect patterns in this disease. In glaucoma fields with marked damage (n = 46), the most frequent pathological Conformity Index was the index for perimetric nerve fiber bundles.
Recombinant human transforming growth factor beta 2 (TGF-beta 2) was cloned and expressed in E. coli. The protein was isolated from inclusion bodies, renatured and purified to a single component as judged by reversed-phase HPLC. The recombinant TGF-beta 2 was shown to have a biological activity equal to that of native TGF-beta 2 in a fibroblast migration assay. Pure, active recombinant TGF-beta 2 has been crystallized from polyethylene glycol 400. The trigonal crystals of spacegroup P3(1)21 or P3(2)21 have unit cell dimensions of a=b=60.6 A, c=75.2 A and diffract beyond 2.0 A.
A procedure to stain the centromeric region of chromosomes for dual beam flow cytometric analysis is described. Serum from a CREST (Scleroderma syndrome) patient presenting a high titer of anticentromeric antibodies was chosen on the basis of specificity of labeling of cells on slides. The high affinity of the antibodies to centromeres and low binding to chromosomal arms allowed the development of an indirect immunofluorescent labeling procedure using isolated and unfixed chromosomes stabilized by Mg++ ions. Discontinuous Ficoll gradients were used to separate chromosomes from unbound antibodies. With this procedure, chromosome clumping and degradation were minimal. The chromosomes were then stained with the DNA dyes Hoechst 33258 and chromomycin A3, before dual beam flow cytometric analysis. Flow karyotypes, with good chromosome peak resolution, were obtained for both human and hamster chromosomes subjected to the immunolabeling procedure. For quantification of FITC fluorescence due to bound antibody, chromosomes were counterstained with Hoechst only. The FITC intensity of antibody-labeled human and hamster chromosomes were 4-10 and 20 times greater than control chromosomes, respectively. These results suggest that the staining procedure may be suitable for immunolabeling of chromosomes with antibodies recognizing other nuclear proteins and their subsequent quantification by flow cytometry.
Human and swine chromosomes were analyzed separately and as a mix to obtain bivariate flow karyotypes. They were normalized to each other in order to use the human chromosomal DNA content as standard. Our results led to the characterization of the "DNA line" in swine identical to the human "DNA line." Estimation of the DNA content in mega-base pairs of the swine chromosomes is proposed. Chromosomal assignment to the various resolved peaks on the bivariate swine flow karyotype is suggested from the relation between DNA content quantified by flow cytometry and chromosomal size. Swine chromosomes 1, 13, 6, 5, 10, 16, 11, 18, and Y were assigned to peaks A, B, C, K, L, N, O, Q, and Y, respectively. Peaks D and E were assumed to contain chromosomes 2 and 14, but without specific assignment. Similarly, P and M peaks were expected to correspond to chromosomes 12 and 17. Of the remaining chromosomes (3, 7, X, 8, 15, 9, and 4), chromosomes 3, 7, and X, which were assigned previously to peaks F, G, and H, respectively, led us to deduce that chromosomes 15 and 8 belonged to peaks I and J, and chromosomes 9, 4, and X to peak H.
Although there are excellent clinical results from using cerium nitrate plus silver sulphadiazine in the treatment of deep dermal burns in our burn centre, its mechanism of action remains unexplained. We set up a prospective study to establish a hypothesis which could explain the formation of the typical leathery crust. Burns treated with cerium nitrate plus silver sulphadiazine and silver sulphadiazine alone were compared histologically and metallurgically. Application of cerium nitrate plus silver sulphadiazine on deep dermal burns caused superficial connective tissue calcification with few deep dermal changes. Cerium penetration was low and silver was never detected intradermally. The precipitation of cerium and pyrophosphate destroys the calcification inhibitory function of the latter, creates multiple calcification nuclei and leads to superficial dermal calcification. No calcium was detectable in biopsies from silver sulphadiazine-treated wounds. No major toxicological side-effects were seen during this study. The clinical results were favourable, as have been reported previously.
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A standard pig flow karyotype (2N = 38 chromosomes) was defined by standardization of several flow karyotypes obtained from stimulated peripheral blood lymphocytes of normal male and female pigs. Depending on the animals under study, the flow analysis of their chromosome suspensions gave rise to bivariate flow karyotypes comprising from 15 to 17 peaks, of which 11 to 15 represented single chromosomes. The results were used to propose a peak nomenclature. In addition, a male miniature pig lymphoblastoid cell line was characterized by flow cytogenetics. A very high-resolution flow karyotype, in which all peaks but one superimposed on those of the standard karyotype, was obtained. Peaks were assigned for chromosomes X and Y. Analysis of flow karyotypes obtained from translocated t(3,7)(p1.3;q2.1) pigs combined with polymerase chain reaction (PCR) studies of major histocompatibility complex (MHC)-linked sequences on flow-sorted chromosomes allowed identification of peaks 3 and 7 of normal pig chromosomes and of the derivative chromosomes associated with the t(3,7)(p1.3;q2.1) translocation.
Angiotensin converting enzyme (ACE) inhibition has shown promising results in diabetic nephropathy, but long-term results on survival are not available. In a cohort of patients receiving antihypertensive treatment predominantly consisting of beta blockers in combination with diuretics, support for an improved survival has been presented. Addition of ACE inhibition to such a combination treatment may be favorable both due to the suggested renoprotective effects of ACE inhibitors and because diuretics activate the renin-angiotensin system. In 10 insulin-dependent diabetic patients with early diabetic nephropathy [urinary albumin excretion rate (UAE) less than 100o micrograms/min], who were receiving continuous therapy with metoprolol and bendroflumethiazide, a double-blind crossover study with four months addition of ramipril 5 mg (Ramace) and placebo was conducted. UAE (radioimmunoassay) and fractional albumin excretion were significantly reduced after the four months of ramipril administration [UAE: 114.1 x/divided by 1.3 (geometric mean x/divided by confidence factor] versus 174.6 x/divided by 1.2 micrograms/min, 2P less than 0.005). Renal plasma flow (clearance of 131I-hippuran) tended to increase [497 +/- 25 (mean +/- SE) vs. 464 +/- 28 ml/min/1.73 m2, 2P = 0.08], while GFR (125I-iothalamate) stayed unchanged (121 +/- 8 vs. 120 +/- 9 ml/min/1.73 m2). Mean arterial pressure during clearance studies fell moderately (95 +/- 3 vs. 101 +/- 1 mm Hg, 2P less than 0.05) and renal resistance was decreased (2P less than 0.03). ACE activity was suppressed in all patients. Twenty-four-hour ambulatory blood pressure measurements were not significantly different after the two periods (daytime averages: 91 +/- 2 vs. 93 +/- 2, nighttime 80 +/- 2 vs. 84 +/- 3 mm Hg).(ABSTRACT TRUNCATED AT 250 WORDS)
Mx protein, an intracellular protein induced by type I interferons (IFNs), is useful as a marker for the IFN-induced state. It is detectable, for example, in leukocytes of patients undergoing IFN-alpha treatment as well as in patients suffering from viral or autoimmune diseases. For immunizations and standardizations, recombinant human MxA protein was expressed in Escherichia coli and purified from inclusion bodies by several steps of chromatography. Two monoclonal antibodies against nonoverlapping epitopes and specific for human Mx protein were selected to establish a simple two-site immunometric enzyme assay. In addition, a monoclonal antibody also reacting with Mx proteins of other species was identified. Prior to assay, whole blood samples were lysed with a nonionic detergent. The sample was incubated on wells coated with a first monoclonal antibody (1304.5.32) together with a second biotinylated monoclonal (1302.34.16), which, after washing, was revealed by an avidin-alkaline phosphatase system. Limit of detection was 5 ng/ml. In two-thirds of normal blood samples (n = 87), Mx protein levels were below 5 ng/ml; 25 samples (29%) had Mx levels between 5 and 50 ng/ml; and 4 samples (5%) were above 50 ng/ml. No Mx was found in plasma, and the mononuclear cell fraction accounted for the bulk of Mx in blood. In vitro, as determined by flow cytometry, monocytes and lymphocytes accumulated Mx protein for 24 h with similar kinetics and remained at plateau levels for more than 70 h. Monocytes contained around eight times more Mx than lymphocytes. The immunoassay was also suitable for detecting Mx after IFN induction in heparinized blood.
Recombinant human synovial fluid phospholipase A2 (rPLA2) and several variants with N-terminal sequences modified by addition or deletion of one or two amino acid residues (ala or Met; Des-Asn1, Leu2) have been expressed in mammalian cells and in Escherichia coli, respectively, purified to homogeneity, and characterized. The observed values for the molecular mass of rPLA2 and variants are in complete agreement with the predicted values for a correctly folded structure containing seven disulfide bridges. Moreover, the relative proportions of the various types of secondary structures of the variants of rPLA2, as measured by CD spectroscopy, are similar to that found for native porcine pancreatic PLA2, indicating that the recombinant proteins are correctly folded. Enzymatic activities of rPLA2 with modified N-termini decreased to 1.3-0.005% of the activity of the mature rPLA2, emphasizing a key role of the N-terminus for catalytic activity.