Characteristics of atypical Neisseria gonorrhoeae from disseminated and localized infections.

Approximately 6% of 1,200 clinical isolates of Neisseria gonorrhoeae were atypical because they produced smaller than normal colonies on conventioal chocolate agar and fermented glucose weakly. Auxotyping studies indicated that these atypical strains required for growth arginine, uracil, and, in most instances, hypoxanthine. In addition, all of them were susceptible to 0.02 U of penicillin/ml. None of the normal colony isolates, including those susceptible to the same low concentration of penicillin, had the same nutritional characteristics. Atypical strains comprised almost half of the isolates from disseminated infections, but only 5% of those from localized infections. Auxotyping was used to identify the contact of a patient who became reinfected nine times with an atypical gonoccal strain. In addition to its usefulness in such epidemiological studies, this technique has enabled us to distinguish a subgroup of gonococci with apparent increased pathogenicity.     

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

Autosomal glycogenosis of liver and muscle due to phosphorylase kinase deficiency is caused by mutations in the phosphorylase kinase beta subunit (PHKB)

Glycogen storage disease due to phosphorylase kinase deficiency occurs in several variants that differ in mode of inheritance and tissue- specificity. This heterogeneity is suspected to be largely due to mutations affecting different subunits and isoforms of phosphorylase kinase. The gene of the ubiquitously expressed beta subunit, PHKB, was a candidate for involvement in autosomally transmitted phosphorylase kinase deficiency of liver and muscle. To identify such mutations, the complete PHKB coding sequence was amplified by RT-PCR of RNA isolated from blood samples of patients and analyzed by direct sequencing of PCR products. The characterization of mutations was complemented by PCR of genomic DNA. In one female and four male patients, we identified five independent nonsense mutations (Y418ter; R428ter; Y974H+E975ter; Q656ter in two cases), one single-base insertion in codon N421, one splice-site mutation affecting exon 31, and a large deletion involving the loss of exon 8. Although these severe translation-disrupting mutations occur in constitutively expressed sequences of the only known beta subunit gene of phosphorylase kinase, PHKB, they are associated with a surprisingly mild clinical phenotype, affecting virtually only the liver, and relatively high residual enzyme activity of approximately 10%.      

Pgp-1hi T lymphocytes accumulate with age in mice and respond poorly to concanavalin A

Aging is associated with an accumulation of T cells functionally hyporesponsive to the effects of mitogens such as concanavalin A. Recent studies in mice and human have identified surface markers useful for distinguishing antigen-stimulated memory T cells from virgin T cells. In mice, memory T cells within the CD8+ cell population have been shown to express relatively high levels of the cell surface glycoprotein Pgp-1. On the theory that aging might diminish the supply of virgin thymic emigrants without compromising the production of memory T cells, we examined the proportion of Pgp-1hiCD4+ and CD8+ cells in the spleen, blood and lymph nodes of mice of varying age. We found a dramatic (2.5-fold) age-associated increase in the percentage of cells with the Pgp-1hi phenotype. By limiting dilution methods, the frequency of concanavalin A-responsive T cells was found to be significantly reduced in the Pgp-1hi cell pool, whether measured by interleukin 2-dependent proliferation, interleukin 2 production or generation of cytotoxic effectors. Pgp-1hi and Pgp-1lo T cells from young mice proliferate equally well when stimulated by optimal doses of phorbol myristate acetate and ionomycin suggesting that the poor responses to concanavalin A do not simply reflect low viability. Aging leads both to an increase in mitogen-hyporesponsive Pgp-1hi T cells, and also to lower responsiveness of cells in the Pgp-1hi subset.     

Bone marrow in polycythemia vera, chronic myelocytic leukemia, and myelofibrosis has an increased vascularity

Several studies have emphasized the significance of neoangiogenesis for tumor growth and progression, but few have focused on malignant hematological disorders. We studied vascular density and architecture in bone marrow samples of patients with chronic myeloproliferative disease (MPD). Vascular structures were immunostained (for von Willebrand factor/FVIII-RAG, CD 31/PECAM or Ulex europeus I for vessels and for vascular endothelial growth factor, VEGF) in samples from patients with polycythemia vera (PV) (n = 7), chronic myelocytic leukemia (CML) (n = 9), and myelofibrosis (MF) (n = 6) when diagnosed and were compared with normal bone marrow specimens (n = 9). We observed that the mean (+/- SD) vessel count per high-power microscopy field (HPF) was 5.3 (+/- 2.1) in normal bone marrow, 5.9 (+/- 2.1) in PV, 10.8 (+/- 3.2) in CML, and 14.4 (+/- 5.5) in MF (P < 0.001 for CMP and MF versus controls). Confocal microscopy, including three-dimensional reconstructions of the blood vessel architecture, confirmed this increased vessel density and revealed tortuous vessel architecture and increased branching in the MPD, particularly in CML and MF. Furthermore, the number of VEGF-positive bone marrow cells was increased in CML and, particularly, in MF. Numbers of VEGF-positive cells and vessels per HPF correlated significantly (r = 0.41; P = 0. 037). Thus the myeloproliferative diseases PV, CML, and MF exhibit neoangiogenesis that is related to diagnosis.