The use of doxycycline in periodontology. Histologic in vivo study on mice affected by diabetes mellitus.

Delay in wound healing process and periodontal disease are 2 possible complications of diabetes mellitus; in case of periodontitis, surgical therapy is necessary if, due to the anatomical and topographical characteristics of periodontal lesions, it is not technically possible to eliminate all the etiological factors by scaling and root-planing therapy only. This work aimed at analysing the effectiveness of doxycycline hyclate as an accelerator of the healing process in laboratory animals (mice with diabetes mellitus). The results obtained after surgical incision of the oral epithelium in diabetic mice show that the topical application of doxycycline hyclate improves, in terms of reparative response time, the regenerative capacity of tissues analysed. Further studies are suggested to test the topical use of doxycycline hyclate in diabetic patients undergoing a periodontal operation in order to accelerate the wound healing process of the tissues concerned.

The interference of medical radionuclides with occupational in vivo gamma spectrometry.

Radiation workers undergo routine monitoring for the evaluation of external and internal radiation exposures. The monitoring of internal exposures involves gamma spectrometry of the whole body (whole body counting) and measurements of excreta samples. Medical procedures involving internal administration of radioactive radionuclides are widely and commonly used. Medical radionuclides are typically short-lived, but high activities are generally administered, whereas occupational radionuclides are mostly long-lived and, if present, are found generally in relatively smaller quantities. The aim of the present work was to study the interference of some common medical radionuclides (201Tl, 9mTc, 57Co, and 131I) with the detection of internal occupational exposures to natural uranium and to 137Cs. Workers having undergone a medical procedure with one of the radionuclides mentioned above were asked to give frequent urine samples and to undergo whole body and thyroid counting with phoswich detectors operated at the Nuclear Research Center Negev. Urine and whole body counting monitoring were continued as long as radioactivity was detectable by gamma spectrometry. The results indicate that the activity of medical radionuclides may interfere with interpretation of occupational intakes for months after administration.

Uptake of ingested uranium after low "acute intake".

The uptake of uranium, ingested as a soluble compound, was studied by monitoring the uranium level in urine by inductively coupled plasma mass spectrometry and through measurement of an isotopic tracer. The high sensitivity of this method allows measurement of uranium levels in urine samples from each voiding, therefore more detailed biokinetic studies are possible. To simulate low "acute intake," five volunteers with "normal" levels (5-15 ng L(-1)) of uranium in urine ingested a grapefruit drink spiked with 100 microg of uranium (235U/238U = 0.245%) as uranyl nitrate, and the level of uranium in their urine after ingestion was monitored. Two techniques were applied to estimate the extent of exposure: a) uranium levels above the normal level for each volunteer; and b) the deviation from natural isotopic ratio. Results were normalized relative to the creatinine concentration, which served as an indicator of urine dilution, to reduce effects due to diurnal changes. The results clearly indicate that currently accepted bio-kinetic models overestimate the time between ingestion of dissolved uranium and its excretion in urine, the maximum of which was found to be around 6-10 h. The uptake fraction was in agreement with recent studies, i.e., 0.1-0.5% of the ingested uranium for four of the subjects but above 1.5% for the fifth, and well below the 5% reported in International Commission on Radiation Protection Publication 54. Finally, partial results from the isotope dilution study indicate that uranium absorbed through the intestine interchanges with uranium retained in body organs. The time scale of this process is quite short, and the acute exposure led to a minimum in the isotopic ratio within hours, while recovery back to natural abundance due to low chronic exposure takes several days.

Uranium in urine--normalization to creatinine.

"Spot samples" of urine are routinely used to monitor occupational exposure to uranium and other toxic heavy metals, such as mercury, lead, and cadmium. In the present work, it was shown that diurnal variations in the uranium concentration in different urine samples from the same individual could be quite large. However, these variations were in correlation to the creatinine level of the same samples, with values of R = 0.72-0.99, for the five subjects studied here. Thus, it is proposed here that uranium concentrations in "spot" urine samples be expressed in terms of ng uranium g(-1) creatinine rather than ng uranium L(-1). Once the 24-h creatinine level is estimated for the individual based on weight, height and age, the adjusted values can be used for determination of the internal dose of uranium.

Chronic-relapsing experimental autoimmune encephalomyelitis (CR-EAE): treatment and induction of tolerance, with high dose cyclophosphamide followed by syngeneic bone marrow transplantation.

We examined the effect of acute immunosuppression with high dose cyclophosphamide (CY), followed by syngeneic T-cell-depleted bone marrow transplantation (SBMT) on chronic-relapsing autoimmune encephalomyelitis (CR-EAE) induced in SJL/J mice by immunization with mouse spinal cord homogenate (MSCH) in adjuvant. Treatment of mice on day 9 post immunization, before the appearance of clinical signs of the disease, delayed the onset of paralysis, but did not affect its clinical course. Treatment on day 2-3 after the first clinical signs led to complete regression of the disease. During a period of 3 months, only one of the 15 mice treated after the the onset of CR-EAE relapsed, as compared to a total of 21 relapses in the 15 untreated animals. A rechallenge with MSCH in adjuvant on day 78 after immunization induced a severe relapse in all untreated mice, with 78% mortality; in contrast, only 25% of mice treated with CY and SBMT relapsed when similarly rechallenged. Lymphocytes from mice treated with CY and SBMT showed reduced in vitro proliferative responses to myelin basic protein (GMBP) and PPD, even after the rechallenge with MSCH. Our results show that high dose CY for elimination of immunocompetent lymphocytes, followed by SBMT rescue, suppresses CR-EAE and induces tolerance to the immunizing antigens. These results may encourage attempts to apply a similar therapeutic principle in life-threatening human neurological autoimmune diseases.

Development of experimental allergic encephalomyelitis during steroid administration. Outcome of neurological immune-mediated disorders under immunosuppressive therapy.

Glucocorticoids are frequently employed as immunosuppressive agents. Following clinical reports of occurrence and exacerbation of neurological immune-mediated conditions in patients receiving steroids, we studied the course of experimental allergic encephalomyelitis (EAE) in rats receiving methylprednisolone prior to and during disease induction and after the appearance of clinical signs. High methylprednisolone dose given prior to EAE induction significantly increased disease duration and the number of days at maximal disability. When the drug was given prior to and during the induction phase, both moderate and high doses exacerbated the EAE course. In contrast, treatment with methylprednisolone after the onset of clinical disease had a markedly beneficial effect. Thus, time of initiation, dosage and duration of treatment may differentially affect the course of EAE. Moreover, the natural course of a neurological immune-mediated condition may be worsened when it is triggered while the patient is under steroid treatment.

[Experimental cochlear damage by neomycin ear drops].

Damage caused by neomycin sulphate solution, identical in concentration to that in ear drops, was tested on the cochleas of 28 guinea pigs. Neomycin sulphate with dexamethasone sodium, and physiologic salt solutions were also tested. Only neomycin solutions damaged the cochlea, and even after only a single exposure. The damage after a single exposure was to the supporting cells and not to the hair cells. However, because of the importance of the supporting cells for normal hearing and because destruction of supporting cells leads to shedding of hair cells, the resulting functional impairment is similar. As a result of further exposure to neomycin the hair cells themselves are damaged, and there is also loss of cell hair. Damage to either the cells or to the hair causes hearing loss. From our work it is clear that in guinea pigs neomycin is absorbed from the middle ear into the inner ear and damages the cochlea. Increased exposure to neomycin increases the damage.

Trends and developments in radioprotection: the effect of nicotinamide on DNA repair.

Recent studies point to the naturally occurring molecules in expression of radiation damage and in protection. DNA repair was shown to be one of the parameters that can be modified to attain improved protection. The need for a natural compound that can enhance DNA repair in order to improve cellular protection focused our attention on nicotinamide (NA). The effects of addition of NA, a precursor for NAD+ synthesis, on the DNA repair capacity following gamma and ultraviolet irradiations were studied in several repair-proficient and repair-deficient cell lines. The addition of low concentrations of NA (less than 3 mM) resulted in increased repair synthesis in the repair-proficient cells. Addition to repair-deficient cells resulted in decreased repair synthesis. Cells which repair damage from one type of radiation, and not from another, responded accordingly to the presence of NA. However, addition of high concentrations of NA to repair-proficient cells resulted in decreased repair synthesis. Thus, nicotinamide can improve the repair capacity in a concentration-dependent manner, but it clearly requires the existence of functional repair processes.

Inhibition of human lymphocyte stimulation by visible light and phthalocyanine photosensitization: mitogen and wavelength dependency.

Mitogenic stimulation of human peripheral blood lymphocytes is inhibited by phthalocyanine photosensitization using visible light. The mechanism of inhibition was studied using stimulating agents differing in their mode of action. Stimulation by the plant lectin phytohemagglutinin (PHA) was the least sensitive to inhibition by photosensitization, followed by the tumor promoter phorbol myristate acetate (PMA) and the calcium ionophore A23187. Mitogenic stimulation could also be inhibited by light only, but higher fluences were needed. The order of efficacy was blue greater than red greater than green light, and the response to A23187 was least affected, followed by an increased inhibition of the response to PHA and PMA stimulation. Possible targets and mechanisms for these effects are discussed.

Inhibition of growth of Chlamydia trachomatis by the calcium antagonist verapamil.

Treatment of BGM (African Green Monkey kidney) cells with the calcium antagonist Verapamil resulted in a reduced yield of chlamydial infectious particles. The inhibitory effect was concentration-dependent, the maximal effect being achieved at 200 microM-Verapamil, which produced a 99.99% reduction of infectious particle yield. Electron microscopy showed that control Chlamydia trachomatis-infected BGM cells contained typical large inclusions in which most of the particles were elementary bodies, whereas Verapamil-treated infected cells contained small inclusions consisting predominantly of reticulate bodies. The findings indicate a possible therapeutic use of this calcium antagonist as an anti-chlamydial drug.

Renal cortical mitochondrial transport of calcium in chronic uremia.

Calcium overload of tubular cells may occur in uremia, and may be the underlying functional abnormality in the continued deterioration of renal function in chronic renal failure. In order to study this question further, the effect of chronic uremia on the calcium transport properties and respiratory rates was examined in mitochondria (Mi) isolated from the cortex of the remnant kidneys of subtotally nephrectomized rats (SNX) and sham operated controls (C). Plasma calcium concentration was similar in both groups of rats, but a significant hyperphosphatemia was seen in SNX, 8.6 +/- 0.6 mg%, as compared to 7.2 +/- 0.2 mg% in C (P less than 0.001). Mi calcium and phosphate concentrations (nmol/mg protein) were significantly elevated in SNX, 49.9 +/- 7.9 and 35.1 +/- 4.2, respectively, in SNX compared to C, 21.2 +/- 4.2 and 21.4 +/- 2.7, respectively (P less than 0.01). Mi respiratory control ratio and ADP/O were similar in both experimental groups. Kinetic parameters for calcium uptake (Ca2+ concentrations in the medium of 1.25 to 16 microM) revealed initial velocities 1.5-fold higher in SNX Mi than in C. Mi retention of calcium in the presence of medium Ca2+ concentrations up to 500 microM was studied. Calcium retention was reduced in SNX: the Mi were unable to retain calcium at concentrations of 250 microM. The addition of ruthenium red to the medium substantially improved calcium retention by the uremic Mi. Chronic parathyroidectomy did not correct either the increased calcium uptake or the poor retention of uremic Mi.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of DNA strand scissions in HeLa cells by human polymorphonuclear leucocytes activated by Chlamydia trachomatis elementary bodies.

Incubation of human polymorphonuclear leucocytes (HPMN) with Chlamydia trachomatis elementary bodies (EB) or phorbol 12-myristate 13-acetate (PMA) resulted in the production of superoxide anions (.O2-) and hydrogen peroxide (H2O2). Exposure of HeLa cells to EB- or PMA-activated HPMN and to EB alone, for 2 h, resulted in the formation of DNA strand scissions (nicks) in the HeLa cells. The nicks were visualized by incorporation of biotin 11-dUTP with its detection by streptavidin-peroxidase, and quantified by using [3H]dCTP in the in situ nuclear nick-translation reaction. Catalase, and to a lesser extent superoxide dismutase, reduced the amount of nicks induced by the EB- or PMA-activated HPMN. The possible relationship between the activity of PMN in chlamydial infections and the development of chronic diseases is discussed.

Combined action of phthalocyanine photosensitization and gamma-radiation on mammalian cells.

The response of Chinese hamster cells and human lymphocytes to the combined action of photosensitization by chloroaluminium phthalocyanine tetrasulfonate and gamma-radiation was studied using colony-forming ability and [3H]thymidine incorporation following mitogenic stimulation respectively, as endpoints. The action of both treatments was usually additive regardless of the sequence of application. However, in human lymphocytes irradiated at low temperature, the photosensitization interacted synergistically with the subsequent ionizing radiation; in this experiment the initial photosensitization reduced the yield of micronuclei produced by gamma-radiation.

Effect of light fluence rate on mammalian cells photosensitization by chloroaluminium phthalocyanine tetrasulphonate.

Phthalocyanines are photosensitizers evaluated for use in photodynamic therapy of cancer. As such, the dependence of the bioresponse on the light fluence rate may be of clinical importance. The effect of the fluence rate of white light from 0.165 to 3.3 kJ m-2 min-1 was studied in Chinese hamster cells and human lymphocytes, using as endpoints colony-forming ability and inhibition of [3H]thymidine incorporation following mitogenic stimulation and dye-photoactivation, respectively. Using Chinese hamster cells exposed to photoexcited chloroaluminium phthalocyanine tetrasulphonate in PBS solution, cytotoxicity was diminished as the fluence rate was reduced. In human lymphocytes changing the fluence rate between 0.33 and 3.3 kJ m-2 min-1 affected the response in a way similar to that of Chinese hamster cells. Human lymphocytes, when exposed to incremental increasing light fluences, 4 h after a conditioning dose, were able to recover from phthalocyanine-induced photodamage, as evidenced by the reappearance of a shoulder on the dose-effect curve. This recovery process during a protracted light exposure, could explain the reduced sensitivity to phthalocyanine photosensitization, compared to exposure at high fluence rates.

Calcium metabolism in uremic nephrocalcinosis: preventive effect of verapamil.

The aim of the present study was to examine calcium metabolism of the renal cortex in experimental chronic renal failure, together with morphologic criteria of nephrocalcinosis and to determine the effect of chronic verapamil administration on these parameters. In subtotally nephrectomized (SNX) rats 3 weeks after surgery, renal cortical calcium content increased more than two-fold. 45Ca incorporation into renal cortical slices in SNX revealed a 35% increase, associated with a 50% increase in a lanthanum-resistant fraction of 45Ca uptake. Radiocalcium wash-out curves in this group demonstrated abnormal retention of the isotope for up to 30 min of incubation. In contrast, radiocalcium incorporation and wash-out in SNX rats chronically treated with verapamil were similar to that obtained in the sham group. Verapamil administration significantly reduced, but did not normalize, renal cortical calcium content. Von Kossa staining demonstrated the deposition of calcium in the renal parenchyma of SNX rats. Ultrastructurally, it was accompanied by mitochondrial disorganization and calcification, as well as by the tubular basement membrane destruction and mineralization. These morphologic patterns of nephrocalcinosis were significantly ameliorated in SNX rats treated with verapamil. We conclude that chronic verapamil administration results in amelioration of uremic nephrocalcinosis.

X-ray microanalysis of uremic nephrocalcinosis: cellular distribution of calcium, aluminium and silicon in uremic nephrocalcinosis.

Nephrocalcinosis with a deposition of aluminum and silicon has been previously reported in uremic rats. To clarify the origin of these elements thin sections of renal cortex were examined by means of electron microscopy and energy dispersive x-ray microanalysis. Studies were performed on subtotally nephrectomized rats (SNX) and sham-operated control animals. Electron microscopy of proximal tubular epithelial cells in SNX rats unlike control rats revealed severe mitochondrial disorganization; irregularity of the tubular basement membrane which exhibited various inclusions; and lamellar bodies within the brush border membrane. On x-ray microanalysis disorganized mitochondria contained calcium and silicon, while secondary lysosomes displayed aluminum and silicon. The tubular basement membrane of SNX rats, as well as lamellar body-like inclusions within the brush border contained calcium, aluminum and silicon together. It is suggested that lysosomal and mitochondrial interaction in proximal tubular cells of uremic rats may contribute to the deposition of calcium together with aluminum and silicon in the renal parenchyma, resulting in nephrocalcinosis.

Psoralen plus near-ultraviolet light: a possible new method for measuring DNA repair synthesis.

A new method is proposed to inhibit semiconservative DNA synthesis in cultured cells while DNA repair synthesis is being measured. The cells are treated with the DNA-crosslinking agent Trioxalen (4,5,8-trimethylpsoralen) plus near-ultraviolet light, and consequently 99.5% inhibition of replicative DNA synthesis is achieved. Additional DNA-damaging agents induce thymidine incorporation into the double-stranded regions of the DNA. The new method gave results very similar to those obtained with the benzoylated naphthoylated DEAE (BND) cellulose method using three human fibroblast strains, of which one had deficient capacity for DNA repair synthesis following treatment with gamma rays and methyl methanesulfonate. The advantages of the new method are simplicity and rapidity, as well as the high extent to which replicative DNA synthesis is inhibited.

Separation of human lymphocytes on Ficoll-Paque gradients: stimulation of cells and depletion of a concanavalin-A responsive radioresistant subpopulation.

A subpopulation of human lymphocytes separated on Ficoll-Paque gradients showed an ultrastructural phenotype characteristic of stimulated cells. Thymidine incorporation was increased fivefold compared with unseparated (Buffy coat) controls. The Ficoll-Paque separated lymphocytes were more sensitive to gamma radiation than unseparated lymphocytes and showed a decreased capability to undergo transformation in response to concanavalin A. Transformation in response to phytohemagglutinin and pokeweed mitogen was the same as for unseparated control lymphocytes. These results are interpreted as the selective depletion of a Con A-responsive T-cell fraction by Ficoll-Paque separation.