Effects of pulmonary rehabilitation on exercise capacity and disease impact in patients with chronic obstructive pulmonary disease and obesity.

OBJECTIVES: To explore the influence of obesity on outcomes of exercise capacity and disease impact in patients with chronic obstructive pulmonary disease (COPD) in response to pulmonary rehabilitation (PR) and to compare outcomes to those of normal weight and overweight counterparts. DESIGN: Secondary data analysis of clinical database. SETTING: St. James's Hospital, Dublin, Ireland. PARTICIPANTS: 155 participants with a primary diagnosis of COPD who completed a PR programme between 2012 and 2014. MAIN OUTCOME MEASURES: Exercise capacity evaluated using the Six Minute Walk Test (6MWT) and the COPD Assessment Test (CAT) evaluated disease impact. RESULTS: Walking distance in the 6MWT improved significantly [mean difference of 55m (95% CI: 42 to 68; p<0.001)] and similarly [F(2, 92)=1.434, p=0.24] across all BMI categories, although the level of improvement reached clinical significance in the normal/underweight and overweight categories only. Disease impact on the CAT score improved across all body mass index (BMI) classifications by 2.3 points (95% CI: 0.9 to 3.6; p<0.050) which reached clinical significance and did not vary across BMI categories [F(2, 80)=0.534, p=0.58]. CONCLUSION: Exercise capacity and self-report disease impact of individuals with COPD improved similarly in response to PR irrespective of BMI.

Multimarker RT-PCR assay for the detection of minimal residual disease in sentinel lymph nodes of breast cancer patients.

The presence of metastases in lymph nodes is the most powerful prognostic factor in breast cancer patients. Routine histological examination of lymph nodes has limited sensitivity for the detection of breast cancer metastases. The aim of the present study was to develop a multimarker reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the detection of minimal residual disease in sentinel nodes of breast cancer patients. RNA was extracted from 30 sentinel lymph nodes (SLN) obtained from 28 patients, three primary breast cancers (positive controls), three lymph nodes from patients with benign diseases, and peripheral blood lymphocytes of 10 healthy volunteers (negative controls). RT-PCR was performed using the following markers; cytokeratin (CK)-19, NY-BR-1 and mammaglobin B. RT-PCR results were compared to enhanced histopathologic examination and immunohistochemistry (IHC). All three positive controls showed strong PCR amplification for all three markers. None of the 13 negative controls was amplified by any of the three markers. Among the 30 SLN analysed, breast cancer metastases were detected in six SLNs by routine histology, in eight by IHC and in 15 by RT-PCR. We conclude that a multimarker RT-PCR assay probing for NY-BR-1, mammaglobin-B, and CK-19 is more sensitive compared to enhanced pathologic examination. This method may prove to be of value in breast cancer staging and prognosis evaluation.

Cloning and characterization of TNKL, a member of tankyrase gene family.

By serological screening of a breast tumor cDNA library we have identified a novel human gene, tnkl, encoding an ankyrin-related protein with a high degree of similarity to tankyrase, the poly(ADP-ribose)polymerase associated with human telomeres (Smith et al, Science 282: 1484). The tnkl gene maps to chromosome 10, while the tnks gene encoding tankyrase is located on chromosome 8. The predicted 1166-aa protein product of the tnkl gene is 78% identical to human tankyrase and 62% to a putative D. melanogaster protein. Since the proteins have essentially identical domain structures, the corresponding genes form a distinct gene family. The possible link between TNKL and cancer justifies its further functional analysis.

Challenges to the development of antigen-specific breast cancer vaccines.

Continued progress in the development of antigen-specific breast cancer vaccines depends on the identification of appropriate target antigens, the establishment of effective immunization strategies, and the ability to circumvent immune escape mechanisms. Methods such as T cell epitope cloning and serological expression cloning (SEREX) have led to the identification of a number target antigens expressed in breast cancer. Improved immunization strategies, such as using dendritic cells to present tumor-associated antigens to T lymphocytes, have been shown to induce antigen-specific T cell responses in vivo and, in some cases, objective clinical responses. An outcome of successful tumor immunity is the evolution of antigen-loss tumor variants. The development of a polyvalent breast cancer vaccine, directed against a panel of tumor-associated antigens, may counteract this form of immune escape.

Identification of a tissue-specific putative transcription factor in breast tissue by serological screening of a breast cancer library.

Application of SEREX (serological analysis of recombinant tumor cDNA expression libraries) to different tumor types has led to the identification of several categories of human tumor antigens. In this study, the analysis of a breast cancer library with autologous patient serum led to the isolation of seven genes, designated NY-BR-1 through NY-BR-7. NY-BR-1, representing 6 of 14 clones isolated, showed tissue-restricted mRNA expression in breast and testis but not in 13 other normal tissues tested. Among tumor specimens, NY-BR-1 mRNA expression was found in 21 of 25 breast cancers but in only 2 of 82 nonmammary tumors. Structural analysis of NY-BR-1 cDNA and the corresponding genomic sequences in the recently released working draft of human genome indicated that NY-BR-1 is composed of 37 exons and has an open reading frame of 4.0-4.2 kb, encoding a peptide of Mr 150,000-160,000. A bipartite nuclear localization signal motif indicates a nuclear site for NY-BR-1, and the presence of a bZIP site (DNA-binding site followed by leucine zipper motif) suggests that NY-BR-1 is a transcription factor. Additional structural features include five tandem ankyrin repeats, implying a role for NY-BR-1 in protein-protein interactions. NY-BR-1 thus represents a breast tissue-specific putative transcription factor with autoimmunogenicity in breast cancer patients. In addition to NY-BR-1, a homologous gene, NY-BR-1.1, was identified in this study. NY-BR-1.1 shares 54% amino acid homology with NY-BR-1 and also shows tissue-restricted mRNA expression. However, unlike NY-BR-1, NY-BR-1.1 mRNA is expressed in brain, in addition to breast and testis. The exon structure of NY-BR-1.1 remains to be defined. Using human genome database, NY-BR-1 was localized to chromosome 10p11-p12, and NY-BR-1.1 was tentatively localized to chromosome 9.

Humoral immunity to human breast cancer: antigen definition and quantitative analysis of mRNA expression.

The ability of the immune system to recognize structurally altered, amplified or aberrantly expressed proteins can be used to identify molecules of etiologic relevance to cancer and to define targets for cancer immunotherapy. In the current study, ninety-four distinct antigens reactive with serum IgG from breast cancer patients were identified by immunoscreening breast cancer-derived cDNA expression libraries (SEREX). A serological profile was generated for each antigen on the basis of reactivity with allogeneic sera from normal individuals and cancer patients, and mRNA expression profiles for coding sequences were assembled based upon the tissue distribution of expressed sequence tags, Northern blots and real-time RT-PCR. Forty antigens reacted exclusively with sera from cancer patients. These included well-characterized tumor antigens, e.g. MAGE-3, MAGE-6, NY-ESO-1, Her2neu and p53, as well as newly-defined breast cancer antigens, e.g. kinesin 2, TATA element modulatory factor 1, tumor protein D52 and MAGE D, and novel gene products, e.g. NY-BR-62, NY-BR-75, NY-BR-85, and NY-BR-96. With regard to expression profiles, two of the novel gene products, NY-BR-62 and NY-BR-85, were characterized by a high level of testicular mRNA expression, and were overexpressed in 60% and 90% of breast cancers, respectively. In addition, mRNA encoding tumor protein D52 was overexpressed in 60% of breast cancer specimens, while transcripts encoding SNT-1 signal adaptor protein were downregulated in 70% of these cases. This study adds to the growing list of breast cancer antigens defined by SEREX and to the ultimate objective of identifying the complete repertoire of immunogenic gene products in human cancer (the cancer immunome).

Activities of counsellors in a hospice/palliative care environment.

This study examined activities related to the provision of psychosocial care by counsellors in the hospice/palliative care setting. A qualitative design using written reports was used in an urban Canadian hospice/palliative care program. A convenient sample of 13 counsellors indicated the activities they typically performed in their work with patients and families. Thematic analysis of the activities directly related to patient and family care was performed and then validated by presenting these activities back to the counsellors in a group setting. Seven themes resulted: 1) companioning; 2) psychosocial assessment, planning, and evaluation; 3) counselling interventions; 4) facilitation and advocacy; 5) patient and family education; 6) consultation and reporting; and 7) team support. These thematic findings confirmed those of previous studies and also highlighted two additional findings. Team support was seen as an activity that directly affected client care, and there was a strong emphasis on the activity of companioning the dying and their families. Also discussed are implications of these results, as well as suggestions for further research.

A recessive contiguous gene deletion causing infantile hyperinsulinism, enteropathy and deafness identifies the Usher type 1C gene.

Usher syndrome type 1 describes the association of profound, congenital sensorineural deafness, vestibular hypofunction and childhood onset retinitis pigmentosa. It is an autosomal recessive condition and is subdivided on the basis of linkage analysis into types 1A through 1E. Usher type 1C maps to the region containing the genes ABCC8 and KCNJ11 (encoding components of ATP-sensitive K + (KATP) channels), which may be mutated in patients with hyperinsulinism. We identified three individuals from two consanguineous families with severe hyperinsulinism, profound congenital sensorineural deafness, enteropathy and renal tubular dysfunction. The molecular basis of the disorder is a homozygous 122-kb deletion of 11p14-15, which includes part of ABCC8 and overlaps with the locus for Usher syndrome type 1C and DFNB18. The centromeric boundary of this deletion includes part of a gene shown to be mutated in families with type 1C Usher syndrome, and is hence assigned the name USH1C. The pattern of expression of the USH1C protein is consistent with the clinical features exhibited by individuals with the contiguous gene deletion and with isolated Usher type 1C.

Serological cloning of a melanocyte rab guanosine 5'-triphosphate-binding protein and a chromosome condensation protein from a melanoma complementary DNA library.

Characterization of immunogenic human melanoma antigens has been a major focus of tumor immunologists over the past two decades, and a broad array of antigens recognized by antibodies and T cells in the autologous host has been defined. In the present study, a melanoma library was screened by SEREX (serological analysis of cDNA expression libraries), and 43 genes were isolated, 2 of which, NY-MEL-1 and NY-MEL-3, encode novel gene products with differential tissue expression. NY-MEL-1 encodes a new rab GTP-binding protein, rab38. Among >40 rab proteins, rab38 has a unique COOH terminus which would allow posttranslational farnesylation and palmitoylation, lipid modifications normally occurring in ras proteins but not in other rab proteins. It is also the only rab gene showing a predominant mRNA expression in melanocytes, a cell-specific expression pattern likely related to melanosomal transport and docking. Northern blot analysis showed no detectable expression in other normal tissues. Consistent with this lineage specificity, rab38 mRNA is expressed in 80-90% of melanoma (17 of 19), but rarely in nonmelanocytic malignancies (1 of 16). The second novel gene isolated, NY-MEL-3, encodes a mitotic protein highly homologous to the Xenopus chromosome condensation protein XCAP-G, designated hCAP-G. Analysis of hCAP-G mRNA expression showed highest expression in the testis among normal tissues and variable expression in tumor cells, reflecting the proliferative activity in these cells. This mitosis-related expression suggests hCAP-G as a possible proliferation marker and a potential prognostic indicator in cancer. These findings provide further support that SEREX can define biologically significant molecules in cancer.

Serological identification of embryonic neural proteins as highly immunogenic tumor antigens in small cell lung cancer.

Serological analysis of expression cDNA libraries (SEREX) derived from two small cell lung cancer (SCLC) cell lines using pooled sera of SCLC patients led to the isolation of 14 genes, including 4 SOX group B genes (SOX1, SOX2, SOX3, and SOX21) and ZIC2. SOX group B genes and ZIC2 encode DNA-binding proteins; SOX group B proteins regulate transcription of target genes in the presence of cofactors, whereas ZIC2 is also suspected to be a transcriptional regulator. These genes are expressed at early developmental stages in the embryonic nervous system, but are down-regulated in the adult. Although SOX2 mRNA can be detected in some adult tissues, ZIC2 is expressed only in brain and testis, and SOX1, SOX3, and SOX21 transcripts are not detectable in normal adult tissues. Of SCLC cell lines tested, 80% expressed ZIC2 mRNA, and SOX1, SOX2, and SOX3 expression was detected in 40%, 50%, and 10%, respectively. SOX group B and ZIC2 antigens elicited serological responses in 30-40% of SCLC patients in this series, at titers up to 1:10(6). In sera from 23 normal adults, no antibody was detected against SOX group B or ZIC2 proteins except for one individual with low-titer anti-SOX2 antibody. Seroreactivity against SOX1 and 2 was consistently higher titered than SOX3 and 21 reactivity, suggesting SOX1 and/or SOX2 as the main antigens eliciting anti-SOX responses. Although paraneoplastic neurological syndromes have been associated with several SCLC antigens, neurological symptoms have not been observed in patients with anti-SOX or anti-ZIC2 antibodies.

Expression of cancer-testis antigens in lung cancer: definition of bromodomain testis-specific gene (BRDT) as a new CT gene, CT9.

In an effort to define new cancer-testis (CT) genes, we investigated whether BRDT, a testis-restricted member of the RING3 family of transcriptional regulators, is also expressed in cancer. Standard RT-PCR expression analysis detected BRDT transcripts in 12 of 47 cases of non-small cell lung cancer and single cases of both squamous cell carcinoma of the head and neck (1/12) and esophagus (1/12) but not in melanoma or in cancers of the colon, breast, kidney and bladder. Typing of 33 non-small cell lung cancers for coexpression of a panel of CT antigens revealed a high incidence (60%) of MAGE-3 mRNA expression, followed by MAGE-1 (36%), CT7/MAGE-C1 (30%), CT10 (30%), SSX4 (23%), BRDT (21%), NY-ESO-1 (21%) and HOM-MEL-40/SSX2 (15%). The coexpression pattern of these antigens provides a foundation for developing a polyvalent lung cancer vaccine.

CT10: a new cancer-testis (CT) antigen homologous to CT7 and the MAGE family, identified by representational-difference analysis.

Assays relying on humoral or T-cell-based recognition of tumor antigens to identify potential targets for immunotherapy have led to the discovery of a significant number of immunogenic gene products, including cancer-testis (CT) antigens predominantly expressed in cancer cells and male germ cells. The search for cancer-specific antigens has been extended via the technique of representational-difference analysis and SK-MEL-37, a melanoma cell line expressing a broad range of CT antigens. Using this approach, we have isolated CT antigen genes, genes over-expressed in cancer, e. g., PRAME and KOC, and genes encoding neuro-ectodermal markers. The identified CT antigen genes include the previously defined MAGE-A6, MAGE-A4a, MAGE-A10, CT7/MAGE-C1, as well as a novel gene designated CT10, which shows strong homology to CT7/MAGE-C1 both at cDNA and at genomic levels. Chromosome mapping localized CT10 to Xq27, in close proximity to CT7/MAGE-C1 and MAGE-A genes. CT10 mRNA is expressed in testis and in 20 to 30% of various human cancers. A serological survey identified 2 melanoma patients with anti-CT10 antibody, demonstrating the immunogenicity of CT10 in humans.

Effect of moderate alcohol upon obstructive sleep apnoea.

Moderate-to-large quantities of alcohol are known to aggravate severe obstructive sleep apnoea (OSA), however, the reported effects of moderate alcohol consumption upon mild-to-moderate OSA are inconsistent. Given the reported benefits of moderate alcohol consumption on cardiovascular mortality, recommendations regarding the management of patients with OSA are difficult to formulate. The aim of this study was to evaluate the effects of moderate alcohol on sleep and breathing in subjects with mild-to-moderate OSA. Twenty-one male volunteers, who snored habitually, underwent polysomnography with and without 0.5 g alcohol x kg body weight (BW)(-1) consumed 90 min prior to sleep time, in random order. The mean blood alcohol concentration (BAC) following alcohol at the time of lights out was 0.07 g x dL(-1). The distribution amongst the various sleep stages was not significantly altered by alcohol. The mean apnoea/hypopnoea index rose from 7.1+/-1.9 to 9.7+/-2.1 events x h(-1) (mean+/-SEM, p=0.017); however, there was no significant change in the minimum arterial oxygen saturation measured by pulse oximetry Sp,O2, apnoea length or snoring intensity. Mean sleep cardiac frequency rose significantly from 53.9+/-1.4 to 59.9+/-1.9 beats x min(-1) (P<0.001) and overnight urinary noradrenalin increased from 14.9+/-2.3 to 18.8+/-2.3 nmol x mmol creatinine(-1) (p=0.061) on the alcohol night compared to the nonalcohol night. To conclude, modest alcohol consumption, giving a mean blood alcohol concentration of 0.07 g x dL(-1), significantly increases both obstructive sleep apnoea frequency and mean sleep cardiac frequency.

Antigens recognized by autologous antibody in patients with renal-cell carcinoma.

The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) is a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. Sixty-five distinct antigens (NY-REN-1 to NY-REN-65) reactive with autologous IgG were identified by SEREX analysis of 4 renal cancer patients and were characterized in terms of cDNA sequence, mRNA expression pattern, and reactivity with allogeneic sera. REN-9, -10, -19, and -26 have a known association with human cancer. REN-9 (LUCA-15) and REN-10 (gene 21) map to the small cell lung cancer tumor suppressor gene locus on chromosome 3p21.3. REN-19 is equivalent to LKB1/STK11, a gene that is defective in Peutz-Jeghers syndrome and cancer. REN-26 is encoded by the bcr gene involved in the [t(9:22)] bcr/abl translocation. Genes encoding 3 of the antigens in the series showed differential mRNA expression; REN-3 displays a pattern of tissue-specific isoforms, and REN-21 and REN-43 are expressed at a high level in testis in comparison to 15 other normal tissues. The other 62 antigens were broadly expressed in normal tissues. With regard to immunogenicity, 20 of the 65 antigens reacted only with autologous sera. Thirty-three antigens reacted with sera from normal donors, indicating that their immunogenicity is not restricted to cancer. The remaining 12 antigens reacted with sera from 5-25% of the cancer patients but not with sera from normal donors. Seventy percent of the renal cancer patients had antibodies directed against one or more of these 12 antigens. Our results demonstrate the potential of the SEREX approach for the analysis of the humoral immune response against human cancer.

Isoforms of the human PDZ-73 protein exhibit differential tissue expression.

Patients with renal and colon cancer frequently develop IgG autoantibodies toward the NY-CO-38/PDZ-73 antigen, a protein of 652 amino acids (73 kDa) which contains three copies of the PDZ protein-protein interaction domain. The gene encoding PDZ-73 mapped to chromosome 11p15.4-p15.1. Additional tissue-specific isoforms were identified: PDZ-45, which lacks the third PDZ domain and the putative PEST protein degradation motif, is expressed in kidney, colon, small intestine, brain and testis; PDZ-54 and PDZ-59, which also lack the third PDZ domains, have unique carboxyl terminal amino acids and are expressed in brain, kidney, bladder, colon cancer and renal cancer; and a putative PDZ-37 isoform, containing only the third PDZ domain, that is expressed in the central nervous system. Immunohistochemical staining with anti-PDZ 73 monoclonal antibodies showed strong cytoplasmic reactivity in epithelial cells of the small intestine, colon and kidney tubules, with a prominent apical staining pattern in cells of the small intestine. The reactivity pattern of the antibodies with various tissues correlated with the mRNA expression pattern of the PDZ-45 isoform. The existence of multiple PDZ-73 isoforms with variations in tissue distribution, PDZ domains, protein degradation sequences and carboxyl terminal structure indicate that these isoforms have distinct tissue-specific functions.

Cancer-testis antigens and ING1 tumor suppressor gene product are breast cancer antigens: characterization of tissue-specific ING1 transcripts and a homologue gene.

SEREX (serological analysis of recombinant tumor cDNA expression libraries) has been applied to several different tumor types and has led to the identification of a wide range of tumor antigens. In this study, a breast cancer library and a normal testicular library were analyzed using autologous and allogeneic breast cancer sera. Thirty genes were isolated, including 27 known genes and 3 previously unknown genes. Among the known genes, two cancer-testis (CT) antigens, NY-ESO-1 and SSX2, previously defined by SEREX analysis, were found. In addition, ING1, a candidate breast cancer suppressor gene, was isolated. This ING1 gene product was also recognized by 2 of 14 allogeneic sera from breast cancer patients but not 12 normal adult sera. Comparison of ING1 cDNA from normal and tumor tissues showed no mutation in the index breast cancer case and revealed the presence of at least three different mRNA transcripts with variable transcription initiation sites and exon usage. Tissue-specific expression of these transcripts was found in normal tissues and tumor cell line mRNAs. Furthermore, a novel gene, designated as ING2, sharing 76% nucleotide homology with ING1 was identified in the breast cancer cDNA library. The basis of the immunogenicity of ING1 and the biological role of ING1 and ING2 need further exploration.