Genetic changes in neoplasms arising in congenital melanocytic nevi: differences between nodular proliferations and melanomas

Large congenital melanocytic nevi (CMN) are at an increased risk of developing melanoma. Several forms of secondary proliferations can arise in congenital nevi on rare occasions. Although some of these closely resemble melanoma both clinically and histologically, metastasis is rare. We used comparative genomic hybridization to analyze chromosomal aberrations in different types of proliferations arising in CMN and compared them to typical congenital nevi, clear-cut melanomas arising in congenital nevi, as well as primary cutaneous melanomas that were not associated with a CMN. Cases of CMN and CMN with secondary proliferations were assigned to six groups according to the predominant histological pattern: group I, bland congenital nevi (n = 6); group II, congenital nevi with foci of increased cellularity (n = 4); group III, CMN with a proliferation simulating superficial spreading melanoma in situ (n = 3); group IV, CMN with a proliferation simulating nodular melanoma (n = 9); group V, proliferating neurocristic hamartoma (n = 1); and group VI, melanoma arising in congenital nevus (n = 6). No aberrations were found in groups I to III, whereas seven of nine cases of group IV, and one of one case of group V, showed aberrations. In group IV six of seven cases with aberrations (86%) showed numerical aberrations of whole chromosomes exclusively. This pattern differed significantly from the findings in melanoma that arose within CMN (n = 6), group VI, or independent of CMN (n = 122) in which only 5% showed numerical changes only. The single case in group V showed aberrations similar to melanoma. The finding of frequent numerical chromosomal aberrations in atypical nodular proliferations arising in CMN identifies these as clonal neoplasms with a genomic instability consistent with a mitotic spindle checkpoint defect. This difference compared to the aberration pattern found in melanoma might explain their more benign clinical behavior and may be of diagnostic value in ambiguous cases.     

Plasma RNA viral load in human immunodeficiency virus type 2 subtype A and subtype B infections

Human immunodeficiency virus type 2 (HIV-2) is much less pathogenic than HIV-1, and HIV-2 infection is associated with plasma viral loads significantly lower than those found in HIV-1 infection. We have developed a real-time quantitative PCR method for measuring the HIV-2 RNA load that covers the range of genetic diversity of HIV-2 isolates and that detects extremely low viral loads. Samples from 49 patients were studied. Proviral DNA was first detected and quantified. The strains that were detected were then genotyped: 21 patients were infected with HIV-2 subtype A and 15 patients were infected with HIV-2 subtype B; 1 patient was infected with a highly divergent strain. Env PCR failed for the remaining 12 patients, so subtypes could not be determined. For viral RNA quantification, a stock of HIV-2 strain NIHZ, which was counted by electron microscopy, was used as the standard. Several primer sets targeting the highly conserved gag region were evaluated. Various primer combinations failed to amplify subtype B strains. With the final primer pair selected, which detected both subtype A and subtype B strains, the sensitivity of the assay was 100% at a viral load of 250 copies/ml and 66% at a viral load of 125 copies/ml. We found a correlation between the CD4(+)-cell count, the clinical stage, and the plasma HIV-2 RNA level. The median plasma HIV-2 RNA value for the 33 asymptomatic patients was 2.14 log(10), whereas it was 3.1 log(10) for the 16 patients with AIDS (P < 0.01). Proviral DNA was detectable in 18 symptom-free patients with high CD4(+)-cell counts, in whom viral RNA was undetectable.     

An evaluation of the inactive mouse X chromosome in somatic cell hybrids

The expression of mouseZfx, Rps4, Ube1x, andXist was evaluated in hamstermouse somatic cell hybrids containing either an active or an inactive mouse X chromosome using polymerase chain reaction of reverse transcribed RNA (RT-PCR). The results showed thatZfx, Rps4, andUbe1x are expressed exclusively from the active mouse X, whileXist is expressed exclusively from the inactive X. These findings confirm the pattern of X inactivation for these mouse genes reported previously based on expression in somatic tissues of F₁ females from interspecific crosses. These results demonstrate the existence of differences between human and mouse X inactivation, as the corresponding human genes,ZFX, RPS4X, andUBE1 escape X inactivation.     

alpha(v)beta(3) Integrin in central nervous system tumors

alpha(v)beta(3) Is an integrin specifically expressed in endothelial cells of newly forming blood vessels. Integrin-mediated angiogenesis is hypothesized to play a central role in the development and the progression of central nervous system neoplasms. Accordingly, it is considered a potential target for antiangiogenic therapy. In the current study, we compare the expression of alpha(v)beta(3) in ependymomas, oligodendrogliomas, pilocytic astrocytomas, medulloblastomas, and vestibular schwannomas (acoustic neuromas). Samples of 5 tumors of each of the 5 tumor types were harvested surgically and frozen. After the pathological diagnosis was confirmed, immunohistochemistry was performed using an anti- alpha(v)beta(3) monoclonal antibody (LM609). The expression of alpha(v)beta(3) was assessed using a 4-tiered (0-3) grading scheme reflecting the percentage of positively staining vessels. All vestibular schwannomas demonstrated strong (grade 3) alpha(v)beta(3) expression. The expression was uniformly prominent in Antoni B regions of the tumors. Of 5 ependymomas, 4 demonstrated uniformly strong alpha(v)beta(3). Oligodendrogliomas, medulloblastomas, and pilocytic astrocytomas demonstrated more variable alpha(v)beta(3). alpha(v)beta(3) may contribute significantly to angiogenesis in vestibular schwannomas and ependymomas. Despite the high vascular density of oligodendrogliomas, pilocytic astrocytomas, and medulloblastomas, these tumors had variable moderate alpha(v)beta(3) expression. This discrepancy suggests temporal and/or regional variability in the angiogenesis in these types of tumor. This study provides the first demonstration of alpha(v)beta(3) expression in vestibular schwannomas, medulloblastomas, and pilocytic astrocytomas.     

Genetic resistance to experimental infection with Mycobacterium bovis in red deer (Cervus elaphus)

Tuberculosis (Tb) caused by Mycobacterium bovis is a worldwide threat to livestock and humans. One control strategy is to breed livestock that are more resistant to Mycobacterium bovis. In a 3-year heritability study 6 farmed red deer stags were selected from 39 on the basis of their differing responses to experimental challenge via the tonsillar sac with approximately 500 CFU of M. bovis. Two stags remained uninfected, two were moderately affected, and two developed serious spreading Tb. Seventy offspring, bred from these six stags by artificial insemination using stored semen, were similarly challenged with M. bovis. The offspring showed patterns of response to M. bovis challenge similar to those of their sires, providing evidence for a strong genetic basis to resistance to Tb, with an estimated heritability of 0.48 (standard error, 0.096; P < 0. 01). This is the first time the heritability of Tb resistance in domestic livestock has been measured. The breeding of selection lines of resistant and susceptible deer will provide an ideal model to study the mechanisms of Tb resistance in a ruminant and could provide an additional strategy for reducing the number and severity of outbreaks of Tb in farmed deer herds. Laboratory studies to identify genetic and immunological markers for resistance to Tb are under way. Preliminary studies showed no associations between NRAMP or DRB genes and resistance to Tb in deer. Patterns of immune responses seen in resistant animals suggest that both innate and acquired pathways of immunity are necessary to produce the resistant phenotype.     

Characterization of the dominant autoreactive T-cell epitope in spontaneous autoimmune haemolytic anaemia of the NZB mouse

NZB mice spontaneously develop autoimmune haemolytic anaemia (AIHA) due to a T helper-dependent autoantibody response against the erythrocyte anion channel protein, Band 3. Here, we characterize the recognition of the Band 3 sequence 861-874, which carries the dominant, I-E(d)-restricted T cell epitope. The ability of N and C-terminal truncated versions of peptide 861-874 to elicit NZB splenic T-cell proliferation indicated that the core epitope spans residues 862-870. Next, a set of alanine substitution analogues was tested to determine which residues functioned either as MHC anchor or TCR contact residues. A combination of proliferation and MHC:peptide binding assays identified residues 862(L), 864(V), 865(L), and 869(K) as I-E(d) anchor residues, and 868(V) as the only TCR contact residue. The ability of the wild-type sequence 861-874 to compete with a high affinity reference peptide for binding to I-E(d) indicates that the escape of pathogenic NZB T cells from purging of the autoreactive repertoire cannot be attributed to ineffective presentation of peptide 861-874 by its restricting element. It will now be possible to design altered peptide ligands of Band 3 861-874, in order to further dissect the mechanisms responsible for the maintenance and loss of T cell tolerance to RBC autoantigens, and to modulate the immune response in AIHA.     

Effects of hormones and lactation on gerbils that seldom scent mark spontaneously

Mongolian gerbils scent mark by rubbing low-lying objects with a ventral scent gland. In males and females that regularly scent mark, the behavior is regulated by gonadal steroid hormones, but the influence of hormones on the scent marking behavior of low- or zero-marking gerbils was unclear. The studies reported here demonstrate that injections of testosterone or of estrogen plus progesterone can stimulate scent marking in some low-marking females but fail to do so in others. Lactation was much more effective for inducing scent marking, but again not all females responded. Although most low- or zero-marking females regularly scent marked while lactating, lactation exaggerated premating differences in marking levels. Very few zero-marking male gerbils exhibited scent marking when castrated and treated with testosterone. In both sexes, scent gland size responded to hormone stimulation even when marking behavior did not.     

HLA-B*35-Restricted CD8+-T-Cell Epitope in Mycobacterium tuberculosis Rv2903c

Few human CD8(+) T-cell epitopes in mycobacterial antigens have been described to date. Here we have identified a novel HLA-B*35-restricted CD8(+) T-cell epitope in Mycobacterium tuberculosis Rv2903c based on a reverse immunogenetics approach. Peptide-specific CD8 T cells were able to kill M. tuberculosis-infected macrophages and produce gamma interferon and tumor necrosis factor alpha.     

Antigen-specific suppression of a primed immune response by dendritic cells mediated by regulatory T cells secreting interleukin-10

Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease. RelB activation is required for myeloid DC differentiation. Here, we show that antigen-exposed DCs in which RelB function is inhibited lack cell surface CD40, prevent priming of immunity, and suppress previously primed immune responses. DCs generated from CD40-deficient mice similarly confer suppression. Regulatory CD4+ T cells induced by the DCs transfer antigen-specific "infectious" tolerance to primed recipients in an interleukin-10-dependent fashion. Thus CD40, regulated by RelB activity, determines the consequences of antigen presentation by myeloid DCs. These observations have significance for autoimmune immunotherapy and suggest a mechanism by which peripheral tolerance might be constitutively maintained by RelB(-) CD40(-) DCs.