The topologic and chronologic patterns of hematopoietic cell seeding in host femoral bone marrow after transplantation.

The early stages of homing, seeding, and engraftment of hematopoietic stem and progenitor cells are poorly characterized. We have developed an optical technique that allows in vivo tracking of transplanted, fluorescent-tagged cells in the host femurs. In this study we used fluorescence microscopy to monitor the topologic and chronologic patterns of hematopoietic cell seeding in the femoral bone marrow (BM) of mice. PKH-labeled cells homed to the femur within minutes after injection into a peripheral vein. Most cells drifted within the marrow space and gradually seeded in clusters close to the endosteal surface of the epiphyseal cortex. Three days after transplantation 85% to 94% (14%) of PKH-labeled cells in the femoral marrow were located within 100 microm of the epiphyseal bone surface (P <.001 versus the more central cells), whereas labeled cells were absent in the femoral diaphysis. Primary seeding of juxtaendosteal, epiphyseal marrow occurred independently of recipient conditioning (myeloablated and nonconditioned hosts), donor-recipient antigen disparity, or the phenotype of the injected cells (whole BM and lineage-negative cells) and was consistently observed in secondary recipients of BM-homed cells. Seeding in regions close to the epiphyseal bone was also observed in freshly excised femurs perfused ex vivo and in femurs assessed without prior placement of optical windows, indicating that the site of primary seeding was not affected by surgical placement of optical windows. Four to 5 days after transplantation, cellular clusters appeared in the more central regions of the epiphyses and in the diaphyses. Centrally located cells showed decreased PKH fluorescence, suggesting that they were progeny of the seeding cells, and brightly fluorescent cells (quiescent first-generation seeding cells) were observed close to the bone surface for as long as 24 days after transplantation. These data indicate that the periphery of the femoral marrow hosts primary seeding and that quiescent cells continue to reside in the periphery for weeks and do not divide. The site of proliferation of transplanted cells is the center of the marrow space.     

Protective actions of human recombinant basic fibroblast growth factor on MPTP-lesioned nigrostriatal dopamine neurons after intraventricular infusion

Basic fibroblast growth factor (bFGF, FGF-2) is a trophic factor for neurons and astrocytes and has recently been demonstrated in the vast majority of dopamine (DA) neurons of the ventral midbrain of the rat. Potential neuroprotective actions of FGF-2 in the l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) model have also been reported. The actions of the FGF-2 have now been further analyzed in a combined morphological and behavioural analysis in the MPTP model of the adult black mouse, using a continuous human recombinant FGF-2 (hrFGF-2) intraventricular (i.v.t.) administration in a heparin-containing (10 IU heparin/ml) mock cerebrospinal fluid (CSF) solution. Tyrosine hydroxylase (TH) immunocytochemistry in combination with computer assisted microdensitometry demonstrated a counteraction of the MPTP-induced disappearance of neostriatal TH-immunoreactive (ir) nerve terminals following the FGF-2 treatment. Unbiased estimates of the total number of nigral TH ir neurons, using stereological methods involving the optical disector (Olympus), showed that the MPTP-induced reduction in the number of nigral TH ir nerve cell bodies counterstained with cresyl violet (CV; by 56%) was partially counteracted by the FGF-2 treatment (by 26%). The behavioral analysis demonstrated an almost full recovery of the MPTP-induced reduction of the locomotor activity after FGF-2 treatment. This action was maintained also 1 week after cessation of treatment. The hrFGF-2 produced an astroglial reaction as determined in the lateral neostriatum and in the substantia nigra (SN) far from the site of the infusion, indicating that the growth factor may have reached these regions by diffusion to activate the astroglia. Immunocytochemistry revealed FGF-2 immunoreactivity (IR) in the nuclei of the astroglia cell population in the dorsomedial striatum and the microdensitometric and morphometric evaluation demonstrated an increase in the number, but not in the intensity, of these profiles on the cannulated side, suggesting the possibility that hrFGF-2 stimulates FGF-2 synthesis in astroglial cells with low endogenous FGF-2 IR. These results indicate that hrFGF-2, directly and/or indirectly via astroglia, upon i.v.t. infusion exerts trophic effects on the nigrostriatal DA system and may increase survival of nigrostriatal DA nerve cells exposed to the MPTP neurotoxin.     

Simultaneous turnover of normal and dysfunctional C1 inhibitor as a probe of in vivo activation of C1 and contact activatable proteases.

Simultaneous turnover of normal and dysfunctional C1-inhibitor (C1-INH) was carried out in 10 normal subjects and 13 patients with rheumatoid arthritis as a measure of the in vivo activation of C1 and the contact activatable enzymes. In the first series of experiments, dysfunctional protein We was used in simultaneous turnover studies in five normal subjects and nine patients. The fractional catabolic rate of the dysfunctional C1-INH, We, (FCR(d)) was unchanged in both groups but the fractional catabolic rate of the normal C1-INH (FCR(n)) was faster in the patients compared to the controls, in particular patients with vasculitis. The enzyme-dependent catabolism defined as FCR(n-d) X concentration of C1-INH X plasma volume, was raised in the patient group, and correlated with disease activity score (r = 0.83, P less than 0.05). Neither FCR(n) nor FCR(d) was dependent on C1-INH concentration. The latter was higher in the patients (206 mg/l compared with 155 mg/l) indicating a very high synthetic rate in the patients (280.81 micrograms/kg/h compared with 179.77 micrograms/kg). In the second series of turnovers in six patients and five normal subjects, another dysfunctional C1-INH, at, was used. The FCR of C1-INH was slower than C1-INH (We) (1.88%/h compared with 2.7%/h). Enzyme-dependent catabolism of C1-INH in these patients were raised and also correlated with disease activity score (r = 0.82, P less than 0.05).     

Long term variation in beam symmetry as a function of gantry angle for a computer-controlled linear accelerator

Testing computer-controlled linear accelerators for patient safety and proper patient dose delivery requires that certain beam characteristics be monitored over an extended period of time. Computer-controlled conformal radiation therapy using asymmetric collimator jaw settings necessitates stable symmetric treatment beams. Long term beam symmetry measurements have been performed on a Philips SL20 dual energy computer-controlled linear accelerator. Symmetry in both the radial and transverse axis of each x-ray beam was monitored for eight gantry positions. These measurements were undertaken to determine the effectiveness of the SL20 beam steering system during dose delivery of 50 monitor units (MU) per field. Evaluation of the data shows that careful beam steering setup procedures result in x-ray beams in which fluctuations in symmetry as a function of gantry angle are within +/- 1.5%. Day to day instabilities produce a total overall variation in beam symmetry on the order of +/- 2.0%. Results suggest the measurement of symmetry as a function of gantry position become a routine quality assurance procedure for this accelerator.     

A nuclear mutant of Chlamydomonas that exhibits increased sensitivity to UV irradiation, reduced recombination of nuclear genes, and altered transmission of chloroplast genes

Meiotic progeny of Chlamydomonas reinhardtii normally receive chloroplast genomes only from the mt+ parent. However, exceptional zygotes, which transmit the chloroplast genomes of both parents or, more rarely, only those of the mt- parent, arise at a low frequency. Mutations at the mt(+)-linked mat-3 locus were found previously to elevate the transmission of chloroplast genomes from the mt- parent, resulting in a much higher than normal frequency of exceptional zygotes. In this paper we demonstrate that an ultraviolet-sensitive nuclear mutation mapping at the uvsE1 locus, which is unlinked to mating type, also promotes chloroplast genome transmission from the mt- parent. This mutant, which was previously shown to reduce recombination of nuclear genes in meiosis, acts synergistically with the mat-3-3 mutation to produce an extremely high frequency of exceptional zygotes. Through the use of restriction fragment length polymorphisms existing in the chloroplast genomes of C. reinhardtii and the interfertile strain C. smithii, we show that chloroplast DNA fragments from the mt- parent normally begin to disappear shortly after zygote formation. However, this process appears to be blocked totally in the absence of wild-type uvsE1 and mat-3 gene products. Our findings are consistent with the hypothesis that both gene products contribute to the mechanism responsible for uniparental inheritance of the chloroplast genome from the mt+ parent.     

Ultrastructural Pathology of Murine Cytomegalovirus Infection in Cultured Mouse Nervous System Tissue

Mouse spinal cord-ganglia cultures were innoculated with murine cytomegalo-virus 14 days after explantation. Intranuclear virus was first observed 4 days after infection. The viruses, which occurred in four forms, were observed in increasing numbers during the ensuing 4 days. Differences were noted in the relative prevalence of certain of these forms in older as compared to younger cultures. This suggests that variations in virus form are related to virus maturation. Cytoplasmic viruses were occasionally observed, but their site of origin is not certain. A variety of cytoplasmic inclusions were seen, particularly in the older cultures. It seems likely that they represent specific cell responses to the presence of the virus. They were not observed in the control cultures, even though some of the latter did show severe degenerative changes.     

Chaetogaster limnaei (Annelida: Oligochaeta) as a parasite of the zebra mussel Dreissena polymorpha,and the quagga mussel Dreissena bugensis (Mollusca: Bivalvia)

Dreissenid mussels, Dreissena polymorpha and D. bugensis, were found to be infected by the naidid oligochaete Chaetogaster limnaei at four sites in the St. Lawrence River. This is the first report of this species infecting dreissenids anywhere in the world. Most worms inhabited the mantle cavity, where they caused erosion of the mantle and gill epithelia as determined by histopathological examination. Others penetrated various tissues; one had invaded the ovary and was feeding on oocytes and ovarian tissues. Of 606 mussels examined, 166 (27.4%) harbored at least 1 C. limnaei. The prevalence varied between 1% and 80%, depending on the collection site and date. The worms were slightly but significantly more prevalent in D. bugensis than in D. polymorpha. The intensity ranged from 1 to 18 worms per infected host. Variations in prevalence and intensity were not related to the size or sex of the host, but the data did suggest some seasonality.