Technetium-99m in the Diagnosis of Thyrotoxicosis

The thyroid uptake at 20 minutes of intravenously administered Technetium-99^m (^99mTc) was measured in 117 patients with a standard scintillation counter. Patients were divided into three groups on the basis of clinical assessment, four-hour ¹³¹I uptake, triiodothyronine (T-3) resin uptake, and protein-bound iodine measurements.In 31 patients with no evidence of thyroid disease the mean ^99m Tc uptake was 1·8% ±S.D. 1·1%. In 32 patients with thyroid enlargement who were euthyroid the mean uptake was 2·5% ±S.D. 2·2%. In 54 thyrotoxic patients the mean uptake was 17·7% with a range of 4·1 to 44%, all cases having an uptake above the upper limit of normal (4·0%). These results agree closely with reported uptake studies using scanning techniques. In seven patients the extrathyroidal neck activity was measured by using a scanner, and the mean was 6·3% of the extrathyroidal total body radioactivity comparing favourably with an assumed 6% used in our calculations.We have shown that the measurement of the thyroid uptake of ^99mTc with a scintillation counter is of value, and that it is not necessary to use scanning techniques in the diagnosis of thyrotoxicosis. Advantages of ^99m Tc are minimal radiation, reduction in patient and laboratory time, and low cost.     

Technetium-99m labeled monoclonal antibodies: Evaluation of reducing agents

We have evaluated five compounds, stannous chloride (SnCl₂), 2-mercaptoethanol (2-ME), dithiothreitol (DTT), dithioerythritol (DTE), and ascorbic acid (AA) to reduce monoclonal antibody MoAb (disulfide groups and compared their efficacy for labeling MoAbs with ^99mTc. The reduction of ^99mTc with dithionite at pH 11 was nearly quantitative. The use of AA, at a molar ratio of 3500:1, for three IgG and three IgM antibodies examined, gave a labeling efficiency greater than 95%. Hence no purification was needed. The immunospecificity of AA preparations determined by specific antigen assay was 84 ± 1% for an IgM and 82.6 ± 1.1% for an IgG, highest among all agents tested. The stability of the tracer was evaluated by challenging the product with such ^99mTc avid agents as cysteine, DTPA, and human serum albumin. By HPLC analysis, no ^99mTc was transchelated using chelating agent to protein molar ratios as high as 500:1. In two separate groups of five mice each, the liver uptake at 4 h post injection averaged 6.8 ± 2.9% per gram for ¹²⁵I-TNT-1 (IgG) and 6 ± 5.1% per gram for the same MoAb labeled with ^99mTc using AA. The AA technique promises to label antibodies with ^99mTc and perhaps with ¹⁸⁶Re, by a simple “kit” procedure.     

Technetium-99m labelled integrated tropane-BAT as a potential dopamine transporter tracer

To reduce the molecular weight of 99mTc-labelled tropanes with the aim to enhance the passage over the blood-brain barrier, a so-called integrated tropane-BAT construct was developed. For this purpose a mercaptoethyl substituent was attached to the amine nitrogen atom of a nortropane precursor and the methyl carboxylate in 2beta-position was converted to a 2-mercaptoethylaminomethylene substituent. This integrated tropane-BAT construct could be labelled efficiently (85-90%) with technetium-99m. Results of LC-MS analysis of the tracer agent support the assumed structure. Biodistribution studies in normal rats (n=3) showed a slightly higher brain uptake for the new tracer agents as compared to 99mTc-TRODAT-1. These results indicate that further biological evaluation of the integrated 99mTc-tropane-BAT is warranted.     

Die Verteilung von Technetium-99m und Jod-131 in der Magenschleimhaut

Zusammenfassung Es wird eine Methode zur Mikrohistoautoradiographie wasserlöslicher Isotope beschrieben und an der Verteilung von ¹³¹J sowie ^99mTc in der Magenschleimhaut demonstriert.Auf Grund mikrohistoautoradiographischer Untersuchungen wird gezeigt, daß Technetium-99m selektiv durch die Belegzellen der Magenschleimhaut ausgeschieden wird. Jod-131 wird demgegenüber von den Hauptzellen des Oberflächenepithels der Magenschleimhaut sezerniert. Die selektive Anreicherung von Technetium-99m in den Belegzellen macht wahrscheinlich, daß Technetium in den Belegzellen eine unmittelbare metabolisch gesteuerte Ausscheidung erfährt. Autoradiographisch wird somit in den Schleimhautzellen nach Gabe von Technetium fast die gesamte Aktivität im Fundus und nur wenig im Antrum gesehen. Die Aktivitätsverteilung in den Lymphgefäßen und Kapillaren der Submucosa ist in Antrum und Fundus gleich.Die Ergebnisse der quantitativen Bestimmung der Aktivitätsverteilung in den verschiedenen Magenabschnitten in Abhängigkeit von der Zeit geben eine gute Bestätigung der mikrohistoautoradiographischen Befunde.

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Distribution of Technetium-99m and Jodine-131 in the gastric mucosaA technique to determine microhistoautoradiographical distribution of water soluble isotopes

Summary A method for microhistoautoradiographic examination of water soluble radioactive isotopes and its use in studying the distribution of ¹³¹J and ^99mTc in the gastric mucosa is described.It is shown that technetium is selectively secreted by the parietal cells of the gastric mucosa. In contrast ¹³¹J is secreted by the chief and mucosal cells. The selective accumulation of ^99mTc in the parietal cells suggests that technetium is secreted via a metabolic pathway. The main activity of ^99mTc is observed in the gastric fundus. The distribution of both isotopes within the submucous lymphoid and capillary vessels of the antrum and fundus is equal.The microhistoautoradiographic results in the different parts of the stomach are confirmed by quantitative scintillation counting.

     

Technetium-99m-labeled antibodies

It is essential in any method for radiolabeling antibody with^99mTc that the labeling procedure is rapid and reliable, producing a highly stable^99mTc-antibody complex with minimal effect on the immunoreactivity of the antibody. In the present study, analysis of the stability and homogeneity of radiolabeled (^99mTc and¹²⁵I) antibodies (HMFG1 and PR1A3) was carried out by fast protein liquid chromatography (FPLC) using superose-6 and S-200 columns, and by polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. Superose 6 and S-200 gel filtration analysis showed the radiolabel (^99mTc or¹²⁵I) eluting with a retention time identical to that of native antibody. No peaks of relative molecular size (Mr) corresponding to possible antibody fragments were seen in either the UV or the radioactive FPLC elution profiles. PAGE analysis of^99mTc labeled antibody, however, revealed the presence of a number of radiolabeled antibody fragments (Mr<IgG) that were not detected by the same analysis of¹²⁵I labeled antibody. The stability of the radiolabeled antibodies in serum in vitro was also studied. FPLC (superose-6) analysis after 45 h incubation in normal serum in vitro revealed 3.3% (HMFG1), and 20% (PR1A3) of the^99mTc on a molecule or aggregate with a Mr greater than that of IgG. There is also the appearance of small amounts of^99mTc-labeled material with a Mr<IgG in the later fractions (2.2% for HMFG1 and 4.9% for PR1A3). Similar results were obtained using radioiodinated antibody, although the small amount of low molecular size material detected as a single peak with a longer retention time than the^99mTc equivalent corresponds to free iodide.     

Technetium-99m autologous phagocyte scanning: a new imaging technique for inflammatory bowel disease.

A method to determine the extent of active inflammatory bowel disease using selective labelling of autologous neutrophils and monocytes by phagocytosis of a technetium-99m (99mTc) stannous oxide colloid is described. Unlike leucocyte scanning techniques using Indium-III (IIIIn), the 99mTc colloid scan uses a cheap, readily available isotope, which specifically labels phagocytes. Scan results in 20 patients with inflammatory bowel disease were compared with barium examinations and colonoscopic appearances. There was close agreement in 15 of 20 patients as to the extent of mucosal disease. In four cases the scan showed more extensive disease than was suggested by barium examination. The scan showed terminal ileal Crohn''s disease in three patients in whom the barium studies of the ileum had been reported as normal. In four patients with inactive disease and normal barium examinations no activity was seen on the scans. The 99mTc phagocyte scan is a sensitive, reliable means of determining the extent of active inflammatory bowel disease and can be used to quantify disease activity.     

Technetium-99m labeled 50H.19 antibody fragments: Interaction of the antibody with platelets

The monoclonal antibody 50H.19 recognized three antigens (M_r = 31-, 40-, 45-K) on normal and thromboasthenic platelets, but only one (M_r = 31-K) on Bernard-Soulier platelets. The intact antibody and its F(ab′)₂ fragments, had direct platelet-aggregating activity, and induced the platelet release reaction. The intact antibody potentiated platelet aggregation induced by platelet-activating factor or thrombin. Additions of indomethacin did not inhibit aggregation: addition of PGI₂, or a calcium channel blocker completely inhibited aggregation. A reduced amount of platelet-aggregating activity was observed with antibody fragments prepared for labeling with ^99mTc by pre-exposure to stannous ions, and herein used in biodistribution studies and elsewhere in thrombus imagining studies (J. Nucl. Med. 27: 1315; 1986). Antibody fragments radiolabeled with ^99mTc bound to isolated platelets and to clots containing platelets.     

Technetium-99m amino acid chelates: correlation of their physico-chemical and physiological parameters. Part I

Ten alpha-aminocarboxylic acids were chelated with 99mTc and purified by sephadex gel chromatography. Their hepatobiliary and renal excretion patterns were determined in rats. It was observed that lipophilicity of these chelates is the only determinant in governing their hepatobiliary excretion, whereas their renal excretion is dependent on some other factors in addition to lipophilicity. Depression of renal excretion in presence of probenecid indicated an interaction of renal tubular transport enzymes with these chelates, which was explained from their hexacoordinated dioxotechnetium (V) chelate structure (II).     

Technetium-99m p-iodophenethyldiaminodithiol (DADT-IPE): Potential brain perfusion imaging agent for SPECT

A new ligand, an N-p-iodophenethyl diaminodithiol (DADT-IPE), an anlog of N-isopropyl-p-iodoamphetamine (IMP), was synthesized and subsequently complexed with ^99mTc, using stannous chloride as a reducing agent. Two complexes (a and b) were separated from ^99mTc-DADT-IPE by high performance liquid chromatography (HPLC). Competitive inhibition studies showed that the IC₅₀ value of DADT-IPE (70 μM) was similar to that of IMP (49 μM). Biodistribution studies of one of the complexes [^99mTc-DADT-IPE(a)] in rats showed that 0.65% of the injected dose of the tracer remained in the brain at 5 min after intravenous injection, with 0.53% of the dose remaining in the brain at 60 min post-injection, whereas the corresponding values for the other complex [^99mTc-DADT-IPE(b)] were 0.34% dose in the brain at 5 min and 0.28% dose in the brain at 60 min post-injection. The half-life for clearance of ^99mTc-DADT-IPE(a) from rat brain was found to be more than 5 h. These results suggested that ^99mTc-DADT-IPE(a) has characteristics which are suitable for cerebral perfusion imaging.     

Nitrogenase of Klebsiella pneumoniae nifV mutants.

The MoFe protein of nitrogenase from Klebsiella pneumoniae nifV mutants, NifV- Kp1 protein, in combination with the Fe protein from wild-type cells, catalysed CO-sensitive H2 evolution, in contrast with the CO-insensitive reaction catalysed by the wild-type enzyme. The decrease in H2 production was accompanied by a stoicheiometric decrease in dithionite (reductant) utilization, implying that CO was not reduced. However, CO did not affect the rate of phosphate release from ATP. Therefore the ATP/2e ratio increased, indicating futile cycling of electrons between the Fe protein and the MoFe protein. The inhibition of H2 evolution by CO was partial; it increased from 40% at pH6.3 to 82% at pH 8.6. Inhibition at pH7.4 (maximum 73%) was half-maximal at 3.1 Pa (0.031 matm) CO. The pH optimum of the mutant enzyme was lower in the presence of CO. Steady-state kinetic analysis of acetylene reduction indicated that CO was a linear, intersecting, non-competitive inhibitor of acetylene reduction with Kii = 2.5 Pa and Kis = 9.5 Pa. This may indicate that a single high-affinity CO-binding site in the NifV- Kp1 protein can cause both partial inhibition of H2 evolution and total elimination of acetylene reduction. Various models to explain the data are discussed.     

The capsular network of Klebsiella pneumoniae.

Attempts at improving chemical fixation for electron-microscopic observation of the capsule of Klebsiella pneumoniae were made. The capsule was preserved by using alcian blue - lanthanum and tris-(1-aziridinyl) phosphine oxide (TAPO) - aldehyde - osmium procedures. Despite the different retention of the overall capsular material and minor variations in morphological details, in both cases the interpretation of ultrastructural patterns suggested that the capsule be composed of a meshed network of thin polysaccharide fibrils radiating from the cell wall. This organization is in keeping with all recognized chemical properties of bacterial polysaccharide capsules or, at least, does not contradict them. Moreover, an effective preservation of bacterial structures other than capsule has been obtained, mostly in specimens fixed by the TAPO-aldehyde-osmium method, a fact which gives further reliability to the technical approach used for capsule visualization.     

Klebsiella pneumoniae in orange juice concentrate.

Fecal coliform-positive, capsule-forming Klebsiella pneumoniae cells were observed in high densities (10(4) to 10(8) CFU/100 ml) in two commercial batches of frozen orange juice concentrate at a cannery in Puerto Rico. Contamination of both lots was gross and included off colors and odors. Isolates of K. pneumoniae from these concentrates revealed growth at 4, 25, and 34 degrees C with generation times from 0.39 to 1.84 h.     

Incorporation of pantothenate into citrate lyase by a pantothenateless mutant of Klebsiella pneumoniae.

A pantothenate-requiring mutant of Klebsiella pneumoniae was isolated. The mutant showed an absolute dependence on pantothenate for growth. When grown in the presence of [14C]pantothenate, the mutant incorporated [14C]pantothenate into citrate lyase (3.4 mol/mol of enzyme). Analysis of a double-labeled enzyme ([14C]pantothenate and [3H]acetate) by gel electrophoresis in sodium dodecyl sulfate showed that both 3H and 14C were associated solely with the smallest subunit, the acyl carrier protein of citrate lyase.     

肺炎克雷伯菌blaCARB-2基因的分布及结构分析

为探讨β-内酰胺酶基因在临床分离肺炎克雷伯菌的分布及其相关可移动遗传元件的结构,本文利用基因组测序、PCR、分子克隆、接合转移和基因组学分析等方法,在对240株临床分离的肺炎克雷伯菌进行混合基因组测序的基础上,着重研究了bla_CARB-2等耐药性基因相关可移动遗传元件的结构及其在肺炎克雷伯菌基因组的定位,克隆获得耐药性质粒携带的若干耐药性基因并测定了它们的功能。结果显示,在240株肺炎克雷伯菌中检出11种β-内酰胺酶基因,其中1株肺炎克雷伯菌(KP1276)被检出bla_CARB-2基因,阳性率为0.42% (1/240);bla_CARB-2位于一个大小为182,450 bp的可接合转移质粒(pKP1276-182),pKP1276-182共编码222个基因,包含7个耐药性基因,分别是bla_CARB-2、bla_KLUC、aadA1、aadA2、cmlA1、dfrA1和sul2。bla_CARB-2是首次在肺炎克雷伯菌中被检出,且bla_CARB-2与其他3个耐药性基因一起构成一个新型结构的1型整合子(int-bla_CARB-2-aadA2-cmlA1-aadA1);对其中的3个耐药性基因bla_CARB-2、aadA2和CmlA1进行了分子克隆和耐药性测定,结果显示它们对相应药物都具有一定的耐药性, 其中bla_CARB-2对青霉素类β-内酰胺药物具有较高的耐药性。本研究结果表明,bla_CARB-2基因已经在肺炎克雷伯菌中出现,其由1型整合子携带编码位于一个可接合转移的质粒上,bla_CARB-2基因有可能在相同及不同种属肠杆菌之间进行水平转移,引起耐药性播散。     

碳青霉烯酶在碳青霉烯类耐药肺炎克雷伯杆菌的分布及分子流行病学

目的了解碳青霉烯酶在碳青霉烯类抗生素耐药肺炎克雷伯杆菌的分布及其分子流行病学。方法收集我院2010年1月至2012年12月分离的碳青霉烯类耐药肺炎克雷伯杆菌120株,采用改良Hodge试验及EDTA协同试验进行碳青霉烯酶表型的确认,用PCR方法扩增碳青霉烯酶基因(KPC、IMP、NDM、VIM、OXA-48),经电泳检测扩增产物后纯化测序。通过MLST及ERIC-PCR进行分子流行病学分析。结果120株碳青霉烯类耐药肺炎克雷伯杆菌中,95株CRKP菌株改良Hodge试验阳性,64株菌株EDTA协同试验阳性。经PCR及测序确认,83株主要携带KPC-2型碳青霉烯酶,占69.2%;其次为IMP-4型金属酶,占28.3%(34/120),NDM-1型金属酶15株,占12.5%,未发现VIM和OXA-48碳青霉烯酶。ERICPCR及MLST分型显示出20个基因分型,其中ST395-A型和ST11-B型主要流行于产KPC-2型菌株中,占43.88%;ST263-B型和ST15-C型主要流行于产NDM-1菌株中,占13.27%;而ST11-B型主要分布于产IMP-4和KPC-2菌株中,占24.49%。结论我院碳青霉烯酶耐药肺炎克雷伯杆菌存在多种碳青霉烯酶,主要以KPC-2为主,产不同种类碳青霉烯酶菌株存在不同的流行基因型。     

肺炎克雷伯菌研究进展

近年来由于各种抗菌药物的广泛使用,导致肺炎克雷伯菌多重耐药现象普遍存在,给临床治疗带来极大的困扰,造成医院获得性肺炎感染严重的现状。着重论述了肺炎克雷伯菌流行现状、耐药机制、致病因子及防治措施。     

Cyclodextrin-Glucanotransferase von Klebsiella pneumoniae

1. When growing with cyclodextrins, Klebsiella pneumoniae M 5 al produces extracellular cyclodextrin glucanotransferase in amounts comparable to those obtained during the growth with potato starch.
2. Intracellular cyclodextrin glucanotransferase-activity was demonstrated to be present in the homogenates of cells grown with cyclodextrins. In addition, an amylomaltase-like enzyme and the maltodextrin phosphorylase could be pointed out. The cyclodextrins are metabolized to glucose-1-phosphate and glucose by the concerted actions of these three enzymes. paraGlucose-1-phosphate is liberated from cyclohexaamylose by the actions of purified cyclodextrin glucanotransferase and purified maltodextrin phosphorylase. The liberation of the sugar phosphate is increased fivefold by addition of glucose as an acceptor. This sugar, however, retards the formation of glucose-1-phosphate from the cyclic compound by the enzymes of the cell extract: In the presence of glucose the amylomaltase is incapable of synthesizing substrates for the phosphorylase from maltose. This experimental result clearly demonstrates that the amylomaltase is involved in the disproportionation of maltosaccharides arising from the cyclodextrins.
3. A NADP⁺-specific glucose dehydrogenase was demonstrated to be present in the cell extracts. This enzyme, which is activated by ADP, may control the energy-depending pool of free glucose. Glucose originates from the disproportionation of maltosaccharides catalyzed by the glucanotransferases.
4. A glucose-1-phosphate-hydrolysing phosphatase, which is shown to be present in the cell extract, seems to be without physiological significance for the metabolism of the cyclodextrins.
5. Preliminary permeation studies make it probable that the cyclodextrins are transported into the cells as such and degraded only within the cells.
6. A scheme for the metabolism of cyclodextrins in Klebsiella pneumoniae M 5 al is proposed.

     

Cyclodextrin-Glucanotransferase von Klebsiella pneumoniae

1. The strain M 5 al of Klebsiella pneumoniae grows excellently with starches. We were able to show that besides the pullulanase associated with the external membrane of the cells the bacterium produces an inducible, extracellular cyclodextrin glucanotransferase [1,4-alpha-D-glucan-4-alpha-(1,4-alpha-glucano)-transferase (cyclising) (EC 2.4.1.19)]. Potato starch and cyclohexaamylose or cycloheptaamylose were found to be the best "inducing" carbon sources for the synthesis of the enzyme. When the bacteria are grown batchwise, maltose is a poorly "inducing" carbon source; larger quantities of the enzyme are synthesized by continuous cultivation with maltose as growth limiting factor. 2. For the determination of the cyclodextrin glucanotransferase-activity an assay method wsa worked out. 3. The enzyme could be separated from the culture filtrate and purified to more than 90% in few steps. At a total yield of 61.2% related to the activity of the culture filtrate employed we received an enzyme solution with the specific activity of 26.6 units/mg protein. Some properties of the enzyme are described. 4. The products formed from amylopectin by the enzyme were analyzed. Somewhat more than half the amylopectin was found as cyclodextrins. 29.3% of the cyclodextrin fraction were cycloheptaamylose, 47.2% cyclohexaamylose and 10.7% exo-branched cyclohexaamylose. 12.8% of cyclohexaamylose were obtained from a cyclodextrin glucanotransferase-limit dextrin after debranching by pullulanase and exposing the product to the action of the glucanotransferase again. 5. The importance of the cyclodextrin glucanotransferase for the utilization of starches by this strain of Klebsiella pneumoniae is discussed. After a first characterization the enzyme is compared to the amylase of Bacillus macerans.