Reconstitution of T-cell repertoire after autologous stem cell transplantation: influence of CD34 selection and cytomegalovirus infection.

The period of immunodeficiency following autologous hematopoietic stem cell transplantation is characterized by transient expansions of CD8+CD45RO+CD57+ T lymphocytes, displaying markers of an activated phenotype. Most evidence suggests that this early reconstitution results from proliferation of mature T cells that have survived conditioning or were transferred with the graft. Although homeostatic mechanisms are thought to act in maintaining total T-cell numbers, the degree to which antigen-driven expansions contribute and the nature of the stimulating antigens remain unclear. CD34 selection of stem cell grafts reduces the available T-cell pool, potentially delaying immune reconstitution and resulting in increased infective complications. In the allogeneic transplantation setting, lymphopenia has been associated with cytomegalovirus (CMV) infection risk and, if persistent, with adverse outcome. We prospectively studied patients undergoing CD34-selected (n = 13) or unselected (n = 13) autologous hematopoeitic stem cell transplantation for immune reconstitution and CMV infection. No significant differences were demonstrated between graft types with respect to lymphocyte subset recovery, T-cell receptor beta-chain variable region spectratype diversity, or CMV DNA detection rates (45% versus 40%). CMV infection was associated with a trend toward higher rather than lower CD8+ counts at 6 weeks posttransplantation (P =.08) that became significant by 3 months (P=.007), and that was associated with decreased T-cell receptor beta-chain variable region spectratype diversity (P =.01). CMV-specific HLA-tetramer analysis demonstrated transient expansions with CDR3 lengths corresponding to those of some of the major posttransplantation T-cell expansions demonstrated by spectratype analysis suggesting that CMV-specific T cells contribute to the pattern of immune reconstitution.     

New Canadian natural health product regulations: a qualitative study of how CAM practitioners perceive they will be impacted

Background  

New Canadian policy to regulate natural health products (NHPs), such as herbs and vitamins were implemented on January 1^st, 2004. We explored complementary and alternative medicine (CAM) practitioners' perceptions of how the new regulations may affect their practices and relationships with patients/consumers.     

CDC group IIc: phenotypic characteristics, fatty acid composition, and isoprenoid quinone content.

Twenty strains of glucose-utilizing, small gram-negative slightly pleomorphic rods that grew well aerobically and that were isolated from clinical specimens formed a phenotypically similar group that was designated CDC group IIc. The phenotypic characteristics of CDC group IIc were most similar to those of CDC groups IIe and IIh, the major differences being that CDC group IIc produced acid from sucrose, hydrolyzed esculin, and usually reduced nitrate. The CDC group IIc strains were analyzed by gas-liquid chromatography for their cellular fatty acid compositions, and all contained relatively large amounts of isobranched hydroxy and nonhydroxy acids. High-performance liquid chromatography and mass spectrometry analysis of the quinone extract showed menaquinone-6 as the major component. Both the cellular fatty acid and isoprenoid quinone compositions were consistent with the profiles of CDC groups IIe and IIh. Thirty percent of the isolates were from human blood.     

Analysis of mycolic acid cleavage products and cellular fatty acids of Mycobacterium species by capillary gas chromatography.

After growth and experimental conditions were established, the mycolic acid cleavage products, constituent fatty acids, and alcohols of representative strains of Mycobacterium tuberculosis, M. smegmatis, M. fortuitum complex, M. kansasii, M. gordonae, and M. avium complex were determined by capillary gas chromatography. Reproducible cleavage of mycolic acid methyl esters to tetracosanoic (24:0) or hexacosanoic (26:0) acid methyl esters was achieved by heating the sample in a high-temperature muffle furnace. The major constituent fatty acids in all species were hexadecanoic (16:0) and octadecenoic (18:1 omega 9-c, oleic) acids. With the exception of M. gordonae, 10-methyloctadecanoic acid was found in all species; moreover, M. gordonae was the only species tested which contained 2-methyltetradecanoic acid. M. kansasii was characterized by the presence of 2,4-dimethyltetradecanoic acid, M. avium complex by 2-eicosanol, and M. tuberculosis by 26:0 mycolic acid cleavage product. The mycolic acid cleavage product in the other five species tested was 24:0. Although a limited number of strains and species were tested, preliminary results indicate that this gas chromatographic method can be used to characterize mycobacterial cultures by their mycolic acid cleavage products and constituent fatty acid and alcohol content.     

Resistance to Leishmania major infection correlates with the induction of nitric oxide synthase in murine macrophages.

Inbred strains of mice differ considerably in their innate resistance to leishmanial infection. BALB/c mice are highly susceptible to cutaneous leishmaniasis caused by Leishmania major, whereas CBA mice are resistant. We now show that this resistance correlates with the ability of macrophages to synthesize nitric oxide (NO) following activation with interferon-gamma or tumor necrosis factor alpha. Furthermore, the larger amounts of NO generated by resistant macrophages are related to higher levels of NO synthase activity, a difference which is not attributable to the number or the affinity of the receptors for interferon-gamma on these cells. The level of NO synthesis by activated macrophages was also correlated to the resistance in a number of other inbred mouse strains tested; macrophages from the resistant B10.S, C57BL and C3H mice produced significantly higher levels of NO than the macrophages from the susceptible BALB.b and DBA/2 mice.     

Cellular fatty acids of Capnocytophaga species.

The cellular fatty acid composition of 18 clinical isolates and 4 reference strains of Capnocytophaga species (Capnocytophaga ochracea, Capnocytophaga gingivalis, and Capnocytophaga sputigena) was determined by gas-liquid chromatography. The fatty acid profiles of the 22 cultures were essentially identical and were characterized by major amounts (60% or greater) of a saturated, iso-branched-chain, 15-carbon acid (13-methyl-tetradecanoate) and the presence of two relatively uncommon saturated, iso-branched, 3-hydroxy acids (13-methyl-3-hydroxy-tetradecanoate and 15-methyl-3-hydroxy-hexadecanoate). The presence and relative amounts of these acids distinguish Capnocytophaga spp. from other gliding bacteria.     

Insertional mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase: evidence that the gene is essential for virus growth

Vaccinia virus encodes a type I DNA topoisomerase whose function in virus replication is not known. To determine whether topoisomerase is required for growth of vaccinia in cell culture, we attempted to isolate null mutations in the topoisomerase gene through insertional mutagenesis. Plasmids containing mutant topoisomerase alleles were constructed by intragenic insertion of the Escherichia coli gpt gene. Recombinant viruses containing the gpt insertion were isolated by selection for growth in the presence of mycophenolic acid. Analysis of the genome structures of drug-resistant viruses revealed that in every case (n = 22) both the wild-type and the gpt-inserted allele were present in viral DNA. We interpret the retention of the wild-type allele as indicative of the essential nature of the topoisomerase gene for vaccinia virus growth.     

The effect of calcium on the maximum velocity of shortening in skinned skeletal muscle fibres of the rabbit

Summary The relationship between maximum shortening velocity (V _max) and free calcium concentration has been studied in skinned single fibres from rabbit psoas and soleus muscles. At both 10 and 15° C,V _max measured in the psoas fibres was found to decrease by 40% when the pCa (-log[Ca²⁺]) was increased from 5.49 (maximally activating) to 6.21. Further decreases inV _max were observed when the pCa was increased to 6.32.V _max measured in soleus fibres at 15° C also decreased when the Ca²⁺ concentration was lowered, though the magnitude of this effect was slightly less than in the psoas fibres. Thus, a distinct effect of Ca²⁺ uponV _max has been shown to occur in mammalian skeletal muscle. The occurrence of this effect in both fast and slow muscle types may indicate that the underlying mechanism in the two cases is similar.     

Fos-like immunoreactivity in brain regions of domestic rams following exposure to rams or ewes

Limbic and basal forebrain-hypothalamic regions from male sheep differing in sexual performance were quantified for fos-like immunoreactivity. Rams classified as high-sexually performing (HP), low-sexually performing (LP), and male-oriented (MO) received noncontact sensory stimulation from either ewes in estrus (HP, n=5; LP, n=4; MO, n=4) or other males (HP, n=5; LP, n=4; MO, n=5) for a 4-h period on each of 3 consecutive days. Following exposure to stimulus animals on the third day, rams were euthanized and their brains were perfused with a 1% paraformaldehyde/1.5% glutaraldehyde solution and sections were analyzed for fos-like immunoreactivity. Brain regions analyzed were the medial amygdala (meAMY), medial preoptic area (mPOA), bed nucleus of the stria terminalis (BNST), and ventromedial hypothalamic nucleus (VMH). Fos-like immunoreactivity differed between groups in the mPOA and BNST but not in the meAMY or VMH. LP rams exposed to estrous ewes had more (P<.05) neurons staining positive for fos and fos-related antigens (FRA) in the mPOA and BNST than LP rams exposed to other rams or MO rams exposed to either estrous ewes or other rams. Numbers of neurons staining positive for FRA in the mPOA and BNST of LP rams exposed to estrous ewes, however, were not different (P>.05) from HP rams exposed to either estrous ewes or other rams. The similar fos-like immunoreactivity in areas important for the display of sexual behavior in HP and LP rams may reflect similar sensory input in these two groups of rams; however, LP rams, in contrast to HP rams, do not appear to respond similarly to the same sensory stimulus.     

Distinctions between endemic and sporadic forms of Epstein-Barr virus-positive Burkitt's lymphoma

Tumour cell lines were established in vitro from 16 cases of Epstein-Barr (EB) virus genome-positive Burkitt's lymphoma (BL), 7 of "endemic" origin (i.e. from holoendemic malarial areas of Africa and of New Guinea) and 9 of "sporadic" origin (i.e. from outside such high-incidence areas). All the BL cell lines thus established were monoclonal by immunoglobulin isotype expression and displayed a characteristic chromosomal translocation, t(8:14) or t(8:22), confirming their malignant origin. Clear differences observed between the individual BL cell lines appeared to be related to their endemic or sporadic status. All 7 endemic cell lines began growth as a carpet of single cells, often with small, loose clumps appearing in later passage. Whilst 3 lines of sporadic origin displayed a similar pattern to the above, the majority of sporadic lines grew as large, tight clumps of cells from the first passage onwards. These differences in growth pattern were reflected by differences in cell surface phenotype, as defined in indirect immunofluorescence tests using a panel of monoclonal antibodies (MAbs) specific for B-lineage-associated antigens. BL cell lines could be classified into 3 separate groups on the basis of their reactivity with 6 particular antibodies (MHM6, AC2, Ki-1, Ki-24, J5 and 38.13). All 7 endemic BL cell lines and 2 of the 3 sporadic BL cell lines which began growth as single cells showed a group-I cell-surface phenotype (MHM6, AC2, Ki-1, Ki-24 negative; J5, 38.13 positive) in early passage. In contrast, all 6 sporadic BL cell lines which began growth in large clumps displayed a distinct group-II phenotype (MHM6, AC2, Ki-1 positive/negative; Ki-24, J5, 38.13 positive); in later passage most of these sporadic lines progressed to a group-III phenotype (MHM6, AC2, Ki-1, Ki-24 positive; J5, 38.13 negative) without loss of those immunoglobulin and chromosomal markers identifying the cells' malignant origin. These clear differences between endemic BL cell lines on the one hand and the majority of sporadic BL cell lines on the other suggest that endemic BL arises from a more restricted range of progenitor B cells than does the sporadic form of the disease.