Human-mouse alignments with BLASTZ

The Mouse Genome Analysis Consortium aligned the human and mouse genome sequences for a variety of purposes, using alignment programs that suited the various needs. For investigating issues regarding genome evolution, a particularly sensitive method was needed to permit alignment of a large proportion of the neutrally evolving regions. We selected a program called BLASTZ, an independent implementation of the Gapped BLAST algorithm specifically designed for aligning two long genomic sequences. BLASTZ was subsequently modified, both to attain efficiency adequate for aligning entire mammalian genomes and to increase its sensitivity. This work describes BLASTZ, its modifications, the hardware environment on which we run it, and several empirical studies to validate its results.     

Enhancement of cALL immunogenicity by co-culture with a CD154 expressing 293 cell line

Pre-B cell acute lymphoblastic leukaemia (cALL) commonly occurs in young patients and although successful conventional therapies are available (such as cytotoxic drugs and bone marrow transplantation) for a proportion of patients (approximately 30%) these are ultimately unsuccessful. Recurrence of disease is a result of the failure of the immune system to recognize these abnormal cells and down-regulation of crucial molecules required for cognate CD4(+) T cell recognition has been postulated as a means of immune escape. In this study we show that an embryonic kidney cell line (293 cells) transfected with CD154 (40 L.1) are capable of not only maintaining the viability of primary ALL cells in culture but can also up-regulate the expression of a number of crucial molecules involved in antigen recognition. We show that 40 L.1 cell stimulation of primary ALL cell cultures can not only enhance the allogeneic and autologous MLR response to such cells but will also induce CTL effectors which are capable of lysing wild-type autologous ALL cells. It is therefore conceivable that such an approach could be used to generate an active anti-tumour response in patients, following conventional therapy, reducing the incidence of recurrence.     

RDCI, the vasoactive intestinal peptide receptor: a candidate gene for the features of Albright hereditary osteodystrophy associated with deletion of 2q37.

Albright hereditary osteodystrophy (AHO) is an autosomal dominant disorder characterised by the presence of brachymetaphalangism, short stature, obesity, and mental retardation. Variable biochemical changes many represent either pseudohypoparathyroidism (PHP) owing to resistance to parathormone (PTH) or pseudopseudohypoparathyroidism (PPHP) with no hormone resistance. In most cases of AHO, reduced levels of Gs alpha have been found and a number of deactivating mutations in the gene for Gs alpha located on chromosome 20q13 have been described. Recently a number of people with an AHO-like phenotype have been reported in whom a deletion of chromosomal region 2q37 has been found in the absence of biochemical abnormalities or a reduction in Gs alpha activity. We present a further female patient with a cytogenetically visible deletion of 2q37, an AHO-like phenotype, and unusual biochemistry suggesting moderate PTH resistance. The vasoactive intestinal peptide receptor (RDCI) has recently been mapped to 2q37 and we propose that this is a candidate gene, hemizygosity of which affects signal transduction and leads to the AHO-like phenotype found in patients with 2q37 deletions.     

Heterogeneity of hepatocyte antigen expression in rat liver carcinogenesis: concordance between neoplastic nodules and tumours.

Fischer F344 rats were given a cyclical diet of 0.06% 2-acetylaminofluorene (AAF), which progressively induced oval cell proliferation, cirrhosis and hyperplastic (or neoplastic) nodules. Primary liver tumours developed from 7 months after ceasing the diet. Liver samples taken during and after AAF administration and specimens of primary tumours were processed into frozen sections and examined microscopically for morphological changes in cell populations, stained histochemically for gamma-glutamyl transpeptidase (GGTase) and four phosphatases, and stained by the immunoperoxidase technique for the presence of antigens detected by seven anti-liver cell monoclonal antibodies and monoclonal antibodies to six oncoproteins. During and after AAF treatment several of the anti-liver antibodies revealed foci of aberrantly or heterogeneously-stained cells, although anti-oncoprotein antibodies showed no consistent changes. Foci of cells positive for GGTase and heterogeneous for adenosine triphosphatase (ATPase) were also seen. Nodules invariably showed heterogeneous antigenicity, raised GGTase and abnormal ATPase expression. Primary tumours exhibited varying degrees of positivity, negativity and heterogeneity with the anti-liver monoclonal antibodies, and all were positive for GGTase. Comparison between various parameters and different lesions showed the greatest concordance between nodules and tumours, suggesting that nodules are probably the precursors of malignant tumours in this system.     

A novel method for selection of invasive tumor cells: Derivation and characterization of highly metastatic K1735 melanoma cell lines based onin vitro andin vivo invasive capacity

Tumor cell invasion of basement membranes is required at several steps in the process of metastasis. To study the genetic and biochemical events mediating invasion, a variant cell line (TK) was selected from the metastatic M2 K1735 murine melanoma cell line. A novel selection procedure was used, based onin vitro andin vivo invasion and growth upon basement membrane and stroma. Additionally, two extrapulmonary metastases of the TK cell line, TK-Eve and TK-Liver, were established as cell lines and characterized. The TK cell line demonstrates greater metastatic potentialin vivo and invasive abilityin vitro than the parent M2 cell line, confirming the validity of the selection procedure. In addition, the M2 and TK cell lines were examined for other cell functions involved in the metastatic process. Cellular growth rates and sensitivity to T lymphocyte and natural killer cell lysis were not determining factors in the metastatic potentials of the M2 and selected cell lines; possible macrophage contribution to metastatic behavior was noted. [³⁵S]methionine pulse labeling of protein synthesis and karyotypic analysis confirm the close relationship of parental and selected cell lines.Supported by contract NDI-23910.Supported by ACS Institution Grant IN-15-Y and NIH Grant MRC-5T34-GM08037.Was a fellow of the Jane Coffin Childs Memorial Fund for Medical Research. This investigation has been aided by a grant from the Jane Coffin Childs Memorial Fund for Medical Research. Also supported by NIH Postdoctoral Fellowship #HD06423.     

Stimulation of neutrophil oxidative metabolism by indomethacin

Human neutrophils exposed to indomethacin demonstrate an enhanced capacity for superoxide ion (O ₂ ^– ) generation when stimulated with opsonized zymosan. Enhancement is not seen with indomethacin-treated cells exposed to solube oxidative stimuli. To further investigate this phenomenon, O ₂ ^– generation, chemiluminescence, and phagocytosis were assessed in human neutrophils preincubated with indomethacin. Zymosan-stimulated O ₂ ^– release was increased from 150 to 300% of controls in neutrophils exposed to 400 g/ml. indomethacin. Enhancement was not reversed by removal of indomethacin from the medium prior to addition of the stimulus and was dose-dependent at drug concentrations of 5 to 400 /ml. Neutrophils exposed to methacin alone also generated more O ₂ ^– than control cells, although this increment was not sufficient to account for the degree of enhancement seen when indomethacintreated cells were exposed to zymosan. Neutrophil cehmiluminescence induced by zymosan was also increased by exposure to indomethacin, and at a drug concentration of 400 g/ml (1.1 mM), enhancement randed from 253 to 333% of controls. As was observed with O ₂ ^– generation, chemiluminescence of neutrophils was increased in the presence of indomethacin alone, although, to a degree far less than was seen when drug-treated cells were stimulated with zymosan. Phagocytosis of radiolabeledS. aureus by neutrophils incubated with indomethacin was increased 13±5% over controls (P<0.01,n=5), but was unaltered by incubation of cells with the buffer used to solubilize the drug. The modest degree of enhancement of phagocytosis suggests that increased particle uptake is not the sole mechanism of oxidative enhancement. The data are in keeping with the hypothesis that indomethacin has a direct effect on the neutrophil plasma membrane and/or the O ₂ ^– -forming oxidase.     

Occurrence of neural tube defects among first-, second-, and third-degree relatives of probands: Results of a United States study

Data on the occurrence of neural tube defects in first-, second-, and third-degree relatives of probands were collected in a United States study. The proportions of affected individuals were 3.2%, 0.5%, and 0.17% respectively. These findings are compared to those from other recent North American studies, and differences are discussed. It is pointed out that accurate recurrence risk figures may not be available, and that caution should be used when counseling families with relatives who are affected with NTD.     

Differential PsaA-, PspA-, PspC-, and PdB-specific immune responses in a mouse model of pneumococcal carriage

Larger numbers of pneumococci were detected in the nasal tract compared to the lung, cervical lymph nodes, and spleen 1, 2, 4, 7, 14, and 21 days after nasal challenge with Streptococcus pneumoniae strain EF3030. In this mouse model of pneumococcal carriage, peripheral S. pneumoniae pneumococcal surface adhesin A (PsaA)-specific humoral responses (immunoglobulin G2a [IgG2a] > IgG1 = IgG2b > IgG3) were significantly higher than pneumococcal surface protein A (PspA)-specific, genetic toxoid derivative of pneumolysin (PdB)-specific, or pneumococcal surface protein C (PspC)-specific serum antibody levels. However, PspA-specific mucosal IgA antibody levels were significantly higher than those against PsaA, PdB, and PspC. In general, both PsaA- and PspA-specific lung-, cervical lymph node-, nasal tract-, and spleen-derived CD4(+) T-cell cytokine (interleukin-4, interleukin-6, granulocyte-macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha) and proliferative responses were higher than those for either PspC or PdB. Taken together, these findings suggest that PsaA- and PspA-specific mucosal responses as well as systemic humoral and T helper cell cytokine responses are predominantly yet differentially induced during pneumococcal carriage.     

Multiplex PCR for direct detection of Shiga toxigenic Escherichia coli strains producing the novel subtilase cytotoxin

We have recently described a novel AB₅ subtilase cytotoxin produced by certain Shiga toxigenic Escherichia coli (STEC) strains. This potentially lethal toxin may contribute to severe gastrointestinal and systemic disease in humans. In this study we have developed a trivalent PCR assay for the detection of the novel toxin A subunit gene subA, as well as stx₁ and stx₂. The three primer pairs used in the assay do not interfere with each other and generate amplification products of 556, 180, and 255 bp, respectively. The assay can be used for determining the toxin genotype of STEC isolates, as well as for direct detection of toxin genes in primary fecal culture extracts.     

Prevalences, genotypes, and risk factors for HIV transmission in South America

HIV cross-sectional studies were conducted among high-risk populations in 9 countries of South America. Enzyme-linked immunosorbent assay screening and Western blot confirmatory testing were performed, and env heteroduplex mobility assay genotyping and DNA sequencing were performed on a subset of HIV-positive subjects. HIV prevalences were highest among men who have sex with men (MSM; 2.0%-27.8%) and were found to be associated with multiple partners, noninjection drug use (non-IDU), and sexually transmitted infections (STIs). By comparison, much lower prevalences were noted among female commercial sex workers (FCSWs; 0%-6.3%) and were associated mainly with a prior IDU and STI history. Env subtype B predominated among MSM throughout the region (more than 90% of strains), whereas env subtype F predominated among FCSWs in Argentina and male commercial sex workers in Uruguay (more than 50% of strains). A renewed effort in controlling STIs, especially among MSM groups, could significantly lessen the impact of the HIV epidemic in South America.