TCRs with high affinity for foreign pMHC show self-reactivity

T cells with high-affinity T cell receptors (TCRs) for a foreign peptide-major histocompatibility complex (pMHC) appear to be negatively selected, even though they have never seen the foreign antigen. To examine how this process operates, we used in vitro yeast display to isolate high-affinity TCRs from the T cell clone 2C. The TCRs showed fast on-rates, which were consistent with reduced CDR (complementarity determining region) flexibility, and cross-reactivity with other cognate pMHCs. T cell hybridomas transfected with a high-affinity TCR were stimulated by endogenous self-pMHC, which suggested that T cells bearing the TCR would be negatively selected. The immune system appears to maintain a repertoire of flexible, low-affinity TCRs at the expense of more effective high-affinity TCRs.     

Let them fly or light them up: matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and fluorescence in situ hybridization (FISH)

This review focuses on clinical bacteriology and by and large does not cover the detection of fungi, viruses or parasites. It discusses two completely different but complementary approaches that may either supplement or replace classic culture-based bacteriology. The latter view may appear provocative in the light of the actual market penetration of molecular genetic testing in clinical bacteriology. Despite its elegance, high specificity and sensitivity, molecular genetic diagnostics has not yet reached the majority of clinical laboratories. The reasons for this are manifold: Many microbiologists and medical technologists are more familiar with classical microbiological methods than with molecular biology techniques. Culture-based methods still represent the work horse of everyday routine. The number of available FDA-approved molecular genetic tests is limited and external quality control is still under development. Finally, it appears difficult to incorporate genetic testing in the routine laboratory setting due to the limited number of samples received or the lack of appropriate resources. However, financial and time constraints, particularly in hospitals as a consequence of budget cuts and reduced length of stay, lead to a demand for significantly shorter turnaround times that cannot be met by culture-dependent diagnosis. As a consequence, smaller laboratories that do not have the technical and personal equipment required for molecular genetic amplification techniques may adopt alternative methods such as fluorescence in situ hybridization (FISH) that combines easy-to-perform molecular hybridization with microscopy, a technique familiar to every microbiologist. FISH is hence one of the technologies presented here. For large hospital or reference laboratories with a high sample volume requiring massive parallel high-throughput testing we discuss matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of nucleic acids, a technology that has evolved from the post-genome sequencing era, for high-throughput sequence variation analysis (1, 2).     

Variable MHC class I engagement by Ly49 natural killer cell receptors demonstrated by the crystal structure of Ly49C bound to H-2K(b)

The Ly49 family of natural killer (NK) receptors regulates NK cell function by sensing major histocompatibility complex (MHC) class I. Ly49 receptors show complex patterns of MHC class I cross-reactivity and, in certain cases, peptide selectivity. To investigate whether specificity differences result from topological differences in MHC class I engagement, we determined the structure of the peptide-selective receptor Ly49C in complex with H-2K(b). The Ly49C homodimer binds two MHC class I molecules in symmetrical way, a mode distinct from that of Ly49A, which binds MHC class I asymmetrically. Ly49C does not directly contact the MHC-bound peptide. In addition, MHC crosslinking by Ly49C was demonstrated in solution. We propose a dynamic model for Ly49-MHC class I interactions involving conformational changes in the receptor, whereby variations in Ly49 dimerization mediate different MHC-binding modes.     

Near-haploid clones in a malignant fibrous histiocytoma

Near-haploid solid tumors are very rare. In a storiform-pleomorphic malignant fibrous histiocytoma (MFH) of bone, we found three cell populations: one with a near-haploid, a second with a near-diploid, and a third with a near-tetraploid chromosome number. The near-haploid cells had few structural rearrangements: i(12p) and t(13q21q) in one clone, and these two and an additional t(19;?)(p11;?) in another clone. One structurally normal copy of all chromosomes was also present, except that the only chromosome 13 was involved in the t(13q21q). There were also two near-diploid clones, one without the t(19;?) and one with a single copy of this derivative chromosome. This is the first MFH reported to have a near-haploid modal chromosome number, and also the first tumor with i(12p) among bone and soft tissue tumors.     

Oxygen metabolites induced by phorbol myristate acetate increase lateral diffusion of wheat germ agglutinin-labeled glycoconjugates in human polymorphonuclear leukocytes

To assess the general effects of protein kinase C (PKC) activation on cell membrane receptor mobility in human neutrophilic polymorphonuclear leukocytes (PMNLs), the lateral diffusion of fluoresceinated succinylated wheat germ agglutinin (S-WGA-FITC)-labeled membrane glycoconjugates was measured using fluorescence recovery after photobleaching (FRAP). Activation of PKC was achieved by incubating the PMNLs with different concentrations (5–100 nM) of phorbol myristate acetate (PMA). The membrane effects of dimethyl sulfoxide (DMSO), another possible membrane perturbant, were also studied. We found that PMA treatment ( 10 nM) increased the glycoconjugate diffusion coefficient (D) 2–2.5-fold. The mobile fraction (R) remained constant, around 30%. With DMSO, no effect on the diffusion was seen. The increase in lateral mobility due to cell stimulation with PMA was totally inhibited by catalase (200 units/ml) but only partly with superoxide dismutase (2000 units/ml). Exogenous hydrogen peroxide (0.01–5 mM) had no effect on glycoconjugate mobility in unstimulated cells. We therefore propose that activation of PKC mediates augmented mobility of glycoconjugate receptors in PMNL, a reaction that seems to be critically dependent on formation of reactive oxygen metabolites. The results indicate that endogenous formation of reactive metabolites upon receptor stimulation may have a general effect on receptor mobility.     

Several chromosomes involved in translocations with chromosome 5 shown with fluorescence in situ hybridization in patients with malignant myeloid disorders

In many patients with myelodysplastic syndromes or acute myeloid leukemia, complex chromosome aberrations can be seen, among which aberrations of chromosome 5 constitute a substantial part. With conventional cytogenetic technique, these aberrations are often identified as deletions or monosomy 5. We analyzed nine patients who, under conventional cytogenetic analysis, showed deletion or monosomy 5. We used fluorescence in situ hybridization with whole-chromosome painting probes to identify the counterpart chromosome and locus-specific identifiers for 5q31 and 5q33 approximately q34. A deletion of 5q was found concomitant with unbalanced translocations. Our results and cases from the literature showed that material from chromosome 5 could be translocated to almost all chromosomes. All patients but one had short survival; this one patient had a preserved 5q31 and 5q33 approximately q34 but a deletion of the q-arm more centromeric than these bands. In eight of the nine patients, further 14 translocations were revealed, not involving chromosome 5.     

Isolation of human immunodeficiency virus (HIV) from plasma during primary HIV infection

Human immunodeficiency virus (HIV) has been isolated from plasma in 6 of 7 patients showing clinical symptoms of a primary HIV infection. Parallel cultures from peripheral blood mononuclear cells (PBMC) yielded virus in 5 patients. In one case, virus could only be isolated from the cerebrospinal fluid but not from peripheral blood. Detectable viremia was transient and preceded the appearance of HIV specific antibodies. After cessation of acute symptoms, the frequency of HIV isolations was similar to that of asymptomatic carriers (23 and 26%, respectively). The role of the immune response in terminating detectable viremia remains to be established.     

Karyotypic evolution in Ph-positive chronic myeloid leukemia in relation to management and disease progression

In a prospective study of 32 patients with chronic myeloid leukemia the frequency of chromosome abnormalities in addition to the Philadelphia chromosome (Ph) increased when the disease progressed. Before metamorphosis, 10 patients (31%) had developed additional abnormalities. Such abnormalities were present in three of them at the time of diagnosis; in the other seven, they were detected late in the chronic phase. New clonal abnormalities heralded or accompanied a more malignant phase of the disorder, usually a blastic leukemia. During metamorphosis, 78% of the patients had additional abnormalities, which in 68% of these cases comprised at least one of +8, +22q- or i(17q). Clones with additional abnormalities disappeared in eight cases, either spontaneously or in association with cytostatic therapy during the chronic or blastic phase. Involvement of chromosome #8, usually in the form of a trisomy, was found in 7 of 12 patients treated with busulfan, but was not found in any of the 10 hydroxyurea-treated patients, of whom 8 were splenectomized early during the chronic phase. Cells from the spleen, obtained by fine needle aspiration or splenectomy were cytogenetically examined in 18 cases during the chronic phase, but abnormalities in addition to the Ph were noted in only one patient, who was examined in the late chronic phase. The same abnormalities were present in bone marrow cells of this patient.