Decreased levels of interleukin-12p40 in the serum of patients with Whipple's disease.

BACKGROUND: An impaired production of interleukin (IL)-12 and T cell interferon-gamma (IFN-gamma) of in vitro stimulated monocytes has been discussed as a pathogenic factor in Whipple's disease (WD). It is unclear whether this defect of cellular immunity is translated to the humoral immune system and to serum correlates. METHODS: We analyzed the serum of 40 patients with Whipple's disease in various degrees of disease activity by sandwich enzyme-linked immunosorbent assay for differences in cytokine and cell adhesion molecule concentrations compared with age- and sex-matched controls. RESULTS: We observed a highly significant reduction of IL-12p40 levels (patients, 0.18+/-0.05 ng/ml (mean+/-SEM); controls, 3.19+/-0.39 ng/ml; p<0.01) in all stages of disease activity, whereas the concentration of IL-12p70 was comparable with controls. Furthermore, we observed a slight decrease in tumour necrosis factor alpha (TNF-alpha) concentrations in the serum of patients (patients, 6.36+/-0.90 pg/ml; controls, 10.5+/-1.23 pg/ml; p<0,05). The levels of other cytokines such as IFN-gamma, IL-2, IL-13 and transforming growth factor beta, as well as soluble cell adhesion molecules lymphocyte function-associated antigen 3 and intercellular adhesion molecule 1, were not significantly different compared with controls. Levels of immunoglobulin G2 (IgG2) measured in the serum of WD patients were below normal in 24 of 29 patients and were even below the 95% confidence interval in 10 patients. CONCLUSION: Our data demonstrate a persistent defect of the cellular immune response with decreased serum concentrations of IL-12p40 and TNF-alpha and decreased IgG2 levels in a large group of WD patients. These data support as in vivo finding the results obtained in previous investigations with stimulated monocytes/lymphocytes. The isolated decrease in IL-12p40 may hint at possible defects in the IL-12/IFN-gamma promoter system.

L523S, an RNA-binding protein as a potential therapeutic target for lung cancer.

Approaches to vaccine-based immunotherapy of human cancer may ultimately require targets that are both tumour-specific and immunogenic. In order to generate specific antitumour immune responses to lung cancer, we have sought lung cancer-specific proteins that can be targeted for adjuvant vaccine therapy. By using a combination of cDNA subtraction and microarray analysis, we previously reported the identification of an RNA-binding protein within the KOC family, L523S, to be overexpressed in squamous cell cancers of the lung. We show here that L523S exhibits significant potential for vaccine immunotherapy of lung cancer. As an oncofetal protein, L523S is normally expressed in early embryonic tissues, yet it is re-expressed in a high percentage of nonsmall cell lung carcinoma. The specificity of L523S expression in lung cancer was demonstrated by both mRNA and protein measurements using real-time PCR, Western blot, and immunohistochemistry analyses. Furthermore, we show that immunological tolerance of L523S is naturally broken in lung cancer patients, as evidenced by detectable antibody responses to recombinant L523S protein in eight of 17 lung pleural effusions from lung cancer patients. Collectively, our studies suggest that L523S may be an important marker of malignant progression in human lung cancer, and further suggest that treatment approaches based on L523S as an immunogenic target are worthy of pursuit.

Detection of mammaglobin in the sera of patients with breast cancer.

Current procedures for the diagnosis of breast cancer are cumbersome and invasive, making detection of this disease difficult. A rapid screening test for early detection of breast cancer would allow for better management of this deadly disease. In this report, we show that, with the exception of the skin, mammaglobin mRNA is specifically expressed in mammary tissue and commonly overexpressed in breast cancer. Mammaglobin is not expressed in other types of cancer including colon, lung, ovarian, and prostate cancer. Breast-specific expression of mammaglobin protein was shown using immunohistochemical methods. Mammaglobin is secreted from both established breast cancer cell lines and primary breast carcinoma cells cultured in vitro. Using a monoclonal antibody-based assay for monitoring the presence of mammaglobin in serum, elevated levels of mammaglobin were detected in sera of patients with breast cancer, but not in healthy women. Thus, mammaglobin, which is overexpressed and secreted from breast carcinoma cells, is detectable in sera of patients with breast cancer and may provide a rapid screening test for the diagnosis and management of breast cancer.

L552S, an alternatively spliced isoform of XAGE-1, is over-expressed in lung adenocarcinoma.

Using a combination of cDNA subtraction and microarray analysis, we report here the identification and characterization of L552S, an over-expressed, alternatively spliced isoform of XAGE-1 in lung adenocarcinoma. Real-time RT-PCR analysis shows that L552S is expressed at levels greater than 10-fold in 12 of 25 lung adenocarcinoma tumors compared with the highest expression level found in all normal tissues tested. L552S is expressed in both early and late stages of lung adenocarcinoma, but it was not detected in large cell carcinoma, small cell carcinoma, or atypical lung neuroendocrine carcinoid. The full-length cDNA for L552S comprises 770 bp and encodes a polypeptide of 160 amino acids. C-terminal 94 amino acids of L552S are identical to a cancer testis antigen, XAGE-1, found in Ewing's sarcoma. Genomic sequence analysis has revealed that L552S and XAGE-1 are alternatively spliced isoforms, and expression of both L552S and XAGE-1 isoforms are present in lung adenocarcinoma. Immunohistochemistry analysis using affinity purified L552S polyclonal antibodies demonstrated specific nuclear staining in 10 of 12 lung adenocarcinoma samples. Furthermore, antibody responses to recombinant L552S protein were observed in seven of 17 lung pleural effusion fluids of lung cancer patients. These results strongly imply that L552S protein is immunogenic and suggest that it might have use as a vaccine target for lung cancer.

Innate resistance to Babesia infection is influenced by genetic background and gender.

Infection of severe combined immunodeficient mice with Babesia sp. strain WA1 was studied to assess the contributions of innate and adaptive immunity in resistance to acute babesiosis. The scid mutation showed little effect in genetically susceptible C3H mice and did not decrease the inherent resistance of C57BL/6 mice to the infection, suggesting that innate immunity plays a central role in determining the course of Babesia infection in these strains. In contrast, the scid mutation dramatically impaired resistance in moderately susceptible BALB/c mice, suggesting that acquired immunity may play an important secondary role. In comparison to their female counterparts, male mice of different genetic backgrounds showed increased resistance to the infection, indicating that the gender of the host may influence protection against babesiosis.

Direct identification of bacteria from positive blood cultures by amplification and sequencing of the 16S rRNA gene: evaluation of BACTEC 9240 instrument true-positive and false-positive results.

In a previous study which evaluated the BACTEC 9240 automated blood culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.), we noted a 1.3% "instrument false-positive" rate. That is, the BACTEC system signaled that a bottle (BACTEC Plus Aerobic/F bottle or BACTEC Anaerobic Lytic/10 bottle) culture was positive but a Gram stain was negative and there was no growth of bacteria or yeasts on subculture to chocolate agar. Furthermore, from the same sample of blood, cultures for fungi using the Isolator blood culture system (Wampole Laboratories, Cranbury, N.J.) were negative for growth. For the present study, we evaluated 76 instrument false-positive samples for the presence of 16S ribosomal DNA using the MicroSeq 500 kit (PE Biosystems, Foster City, Calif.). These samples also were negative for fungi by the Isolator method. This kit has a PCR module and sequencing module for the amplification and sequencing of the 16S RNA gene and provides a database for sequence alignment and identification of bacteria. To optimize the assay, we evaluated the effect of adding 0.5% bovine serum albumin to the sample from blood culture bottles and found that it decreased the effects of inhibitors on the PCR. Two control groups of blood culture specimens were also evaluated. One group (n = 45) were "instrument true positives"; the instrument signaled positive, and subsequent Gram stains were positive and subcultures on chocolate agar grew bacteria. The other group (n = 20) were "instrument true negatives"; the instrument signaled negative, the Gram stain was negative, and subcultures on chocolate agar and from the Isolator tube on fungal media showed no growth. None of the 76 instrument false-positive samples had evidence for 16S rRNA gene sequences. All of the instrument true-positive samples and all of the instrument true-negative specimens were positive and negative, respectively, using the MicroSeq 500 kit. Total peripheral white blood cell counts were statistically significantly higher for patients who had instrument false-positive results than for patients who had instrument true-positive or true-negative results (P = 0.001). We conclude that instrument false positives signaled by the BACTEC 9240 system are not due to bacteria in the blood culture samples.

A first-tier rapid assay for the serodiagnosis of Borrelia burgdorferi infection.

BACKGROUND: The present recommendation for the serologic diagnosis of Lyme disease is a 2-tier process in which a serum sample with a positive or equivocal result by an enzyme-linked immunosorbent assay (ELISA) or immunofluorescent assay is then followed by supplemental testing by Western blot. Our laboratory has developed recombinant chimeric proteins composed of key Borrelia epitopes. These novel antigens are consistent and are easily standardized. METHODS: We adapted these recombinant proteins into a new immunochromatographic format that can be used as a highly sensitive and specific first-tier assay that can be used to replace the ELISA or immunofluorescent assay. RESULTS: This rapid test was equally sensitive (P>.05) and more specific (P<.05) than a frequently used commercial whole cell ELISA. The overall clinical accuracy achieved on agreement studies among 3 Lyme research laboratories on clinically defined serum panels was shown to be statistically equivalent to the commercial ELISA. The assay can detect anti-Borrelia burgdorferi antibodies in either serum or whole blood. CONCLUSION: This sensitive and specific rapid assay, which is suited for the physician's office, streamlines the 2-tier system by allowing the physician to determine if a Western blot is necessary at the time of the initial office visit.

Bartonella henselae serostatus is not correlated with neurocognitive decline in HIV infection.

Bartonella henselae has been implicated as a significant cause of HIV-associated dementia. We attempted to confirm this association by utilizing the database of the San Diego HIV Neurobehavioral Research Center, which collects longitudinal neurocognitive and laboratory data on over 500 HIV-infected participants. Utilizing an immunofluorescent assay we found that 11% of 177 subjects, half of whom had documented neurocognitive decline, were seropositive for B. henselae. There was no correlation between B. henselae seropositivity and neurocognitive decline. The role of B. henselae in HIV-associated dementia remains ambiguous.

Transcriptional complementarity in breast cancer: application to detection of circulating tumor cells.

BACKGROUND: We used a combination of genetic subtraction, silicon DNA microarray analysis, and quantitative PCR to identify tissue- and tumor-specific genes as diagnostic targets for breast cancer. METHODS AND RESULTS: From a large number of candidate antigens, several specific subsets of genes were identified that showed concordant and complementary expression profiles. Whereas transcriptional profiling of mammaglobin resulted in the detection of 70% of tumors in a panel of 46 primary and metastatic breast cancers, the inclusion of three additional markers resulted in detection of all 46 specimens. Immunomagnetic epithelial cell enrichment of circulating tumor cells from the peripheral blood of patients with metastatic breast cancer, coupled with RT-PCR-based amplification of breast tumor-specific transcripts, resulted in the detection of anchorage-independent tumor cells in the majority of patients with breast cancer with known metastatic disease. CONCLUSION: Complementation of mammaglobin with three additional genes in RT-PCR increases the detection of breast cancers in tissue and circulating tumor cells.

Serodiagnosis of human granulocytic ehrlichiosis by using novel combinations of immunoreactive recombinant proteins.

A panel of seven recombinant antigens, derived from Ehrlichia phagocytophila (the agent of human granulocytic ehrlichiosis), was evaluated by class-specific enzyme-linked immunosorbent assays (ELISAs) for utility in the diagnosis of the infection. Fourteen genomic fragments, obtained by serologic expression screening, contained open reading frames (ORFs) encoding 16 immunodominant antigens. Eleven of these antigens were members of the major surface protein (MSP) multigene family. Alignment of their predicted protein sequences revealed a pattern of conserved sequences, which contained short direct repeats, flanking a variable region. In addition, two genomic clones contained two and three MSP ORFs, respectively, indicating that these genes are clustered in tandem copies. The implications for this pattern of both genomic and protein arrangements in antigenic variations of MSPs and in their utilities in a diagnostic assay are discussed. In addition to two MSP recombinant antigens (rHGE-1 and -3) and a fusion protein of these antigens (rErf-1), five further recombinants were evaluated by ELISA. Two of these antigens (rHGE-14 and -15) were novel, while a third (rHGE-2), with no known function, has been described. The final two recombinant antigens (rHGE-9 and -17) represent overlapping segments of the ankyrin gene (ank). The addition of rHGE-9 ELISA data resulted in the detection of 78% (21 of 27) of acute-phase sera. When serologic data for all recombinants are combined, 96.2% (26 of 27) of convalescent-phase patient serum samples and 85.2% (23 of 27) of acute-phase patient serum samples are detected, indicating the potential of these antigens for use in the development of a rapid serologic assay for the detection of E. phagocytophila infection.

Serological expression cloning and immunological evaluation of MTB48, a novel Mycobacterium tuberculosis antigen.

Improved diagnostics are needed for the detection of Mycobacterium tuberculosis, especially for patients with smear-negative disease. To address this problem, we have screened M. tuberculosis (H37Rv and Erdman strains) genomic expression libraries with pooled sera from patients with extrapulmonary disease and with sera from patients with elevated reactivity with M. tuberculosis lysate. Both serum pools were reactive with clones expressing a recombinant protein referred to here as MTB48. The genomic sequence of the resulting clones was identical to that of the M. tuberculosis H37Rv isolate and showed 99% identity to the Mycobacterium bovis and M. bovis BCG isolate sequences. The genomic location of this sequence is 826 bp upstream of a region containing the esat-6 gene that is deleted in the M. bovis BCG isolate. The mtb48 1,380-bp open reading frame encodes a predicted 47.6-kDa polypeptide with no known function. Southern and Western blot analyses indicate that this sequence is present in a single copy and is conserved in the M. tuberculosis and M. bovis isolates tested but not in other mycobacterial species tested, including Mycobacterium leprae and Mycobacterium avium. In addition, the native protein was detected in the cytoplasm, as was a processed form that was also shed into the medium during culture. Serological analysis of recombinant MTB48 and the M. tuberculosis 38-kDa antigen with a panel of patient and control sera indicates that the inclusion of recombinant MTB48 in a prototype serodiagnostic test increases assay sensitivity for M. tuberculosis infection when it is combined with other known immunodominant antigens, such as the 38-kDa antigen.

Gestational attenuation of Lyme arthritis is mediated by progesterone and IL-4.

Infection of different strains of laboratory mice with the agent of Lyme disease, Borrelia burgdorferi, results in arthritis, the severity of which has been correlated with the dominance of Th1 cytokines. In this study, we demonstrate that changes in B. burgdorferi-specific immunologic responses associated with pregnancy can alter the outcome of Lyme arthritis in mice. Whereas nonpregnant female C3H mice consistently developed severe Lyme arthritis, pregnant mice had a marked reduction in arthritis severity that was associated with a slight reduction in IFN-gamma and markedly increased levels of IL-4 production by B. burgdorferi-specific T cells. Similar reductions in arthritis severity and patterns of cytokine production were observed in nonpregnant, progesterone-implanted mice. Ab neutralization of IL-4 in progesterone-implanted mice resulted in severe arthritis. Our results are consistent with the known shift toward Th2 cytokine expression at the maternal-fetal interface, and are the first to show a pregnancy-related therapeutic effect in an infectious model.

Human papillomavirus in cutaneous squamous cell carcinoma and cervix of a patient with psoriasis and extensive ultraviolet radiation exposure.

"High-risk" human papillomaviruses (HPVs) are associated with intraepithelial neoplasia and cancer of the uterine cervix. HPV has also been found in nonmelanoma skin cancer (NMSC), especially in squamous cell carcinomas (SCCs) of immunosuppressed patients. Recently, lesions of psoriasis have been shown to harbor HPV, and patients with psoriasis often have a history of extensive therapy with ultraviolet radiation (UVR). UVR is the major known risk factor in the occurrence of NMSC, in which HPV may be a cofactor for SCC. We report an otherwise healthy, nonimmunosuppressed patient with psoriasis who had a history of extensive exposure to UVR and experienced multiple SCCs on UV-exposed body sites. By the polymerase chain reaction method, we detected HPV in 5 of 9 SCCs. Automated sequencing showed HPV types 12 and 17. Only 1 of 3 normal skin specimens was HPV positive (HPV type 17). This positive specimen was from UV-exposed skin; one of the two HPV-negative, normal skin specimens was located on a body site not exposed to sun. In addition, HPV type 62 was found in a brush specimen of the uterine cervix. This case report suggests an association between psoriasis, HPV infection, and UVR exposure, in onset of SCC.

Tropheryma whippelii DNA is rare in the intestinal mucosa of patients without other evidence of Whipple disease.

BACKGROUND: Little is known about the pathogenesis of Whipple disease, the reservoirs of Tropheryma whippelii, and the proportion of persons harboring the bacterium without "classic" intestinal abnormalities. OBJECTIVE: To assess the presence of T. whippelii in patients undergoing upper endoscopy for a variety of indications. DESIGN: Prospective and routine diagnostic examination of patients. SETTING: Three academic medical centers in California; Minnesota; and Heidelberg, Germany. PATIENTS: 342 patients undergoing endoscopy for evaluation of dyspepsia or possible peptic ulcer (group A, 173 patients), malabsorption (group B, 37 patients), or clinical suspicion of Whipple disease (group C, 132 patients). MEASUREMENTS: Small-intestinal biopsy specimens were tested by polymerase chain reaction for T. whippelii DNA and examined for histopathologic abnormalities. RESULTS: All patients with negative histologic findings also had negative results for T. whippelii DNA. CONCLUSIONS: T. whippelii occurs only rarely in intestinal mucosa that lacks histopathologic evidence of Whipple disease. The human small intestinal mucosa is an unlikely reservoir for this organism.

Characterization of culture-derived spiral bacteria by 16S ribosomal RNA gene sequence analysis.

Broad range amplification and sequence analysis of the 16S ribosomal RNA gene was used to identify three spiral-form organisms. The agents were identified as Campylobacter fetus, "Flexispira rappini", and Borrelia burgdorferi, respectively, using either proprietary or public sequence databases. In each case, the rDNA sequence showed 99-100% homology with known sequence data. Sequence-based analysis for each isolate required only 2-3 days, whereas traditional means of identification took 8-12 days to complete. The identification of spirochetes and vibrio-like agents from human clinical samples is often time consuming and results may be difficult to interpret, sometimes due to atypical phenotypic characteristics. Analysis of 16S rDNA or other molecular targets may provide a way to accurately and rapidly characterize isolates that are recalcitrant to speciation.

Natural history of vancomycin-resistant enterococcal colonization in liver and kidney transplant recipients.

At Mayo Medical Center (Rochester, MN), surveillance rectal (and other-site) cultures have been routinely collected from liver transplant recipients as part of a selective bowel decontamination program. Beginning in 1995, vancomycin-resistant enterococcus (VRE) colonization and infection were identified in Mayo Clinic liver and kidney transplant patients through our surveillance cultures. The purpose of this study is to describe the natural history of VRE colonization in this patient population. Fifty-two patients with VRE colonization (predominantly with a single vanB clone) were identified from September 1995 through December 1997. Five hundred ninety cultures were reviewed for this study (mean, 11.3 cultures/patient). The median time from initial VRE colonization to the last surveillance culture obtained was 306 days (range, 1 to 1,393 days). VRE infection was documented in 6 patients (11.3%). Eighteen patients (35%) met the criteria for clearance of VRE colonization, defined as VRE-negative rectal culture results on at least 3 consecutive occasions greater than 1 week apart. However, VRE was detected on subsequent surveillance cultures from 2 of these patients (11% relapse rate). Of the remaining 34 patients, 16 remained colonized with VRE and 18 did not meet the definition for clearance of VRE colonization because of incomplete follow-up. This study documents that VRE colonization usually persists for months to years in liver and kidney transplant patients.

Human papillomavirus type 16 integrations in cervical tumors frequently occur in common fragile sites.

The development of cervical cancer is highly associated with human papillomavirus (HPV) infection. HPV integration into the genome of infected cervical cells is temporally associated with the acquisition of the malignant phenotype. A relationship between the sites of HPV integration in cervical cancer and the position of the common fragile sites (CFSs) has been observed at the cytogenetic level. To explore this relationship at the molecular level, we used a PCR-based method to rapidly isolate cellular sequences flanking the sites of HPV16 integrations in primary cervical tumors. Human bacterial artificial chromosome clones were isolated based on these flanking sequences and used as probes for fluorescence in situ hybridization on metaphases derived from cells cultured in the presence of aphidicolin. Our data demonstrate that HPV16 integrations in cervical tumors frequently occur within CFSs at the molecular level. In addition, we have determined the precise molecular locations of the CFSs FRA6C and FRA17B.

Differential distribution of sequence variations in HPV-16 E6.

OBJECTIVE: The E6 regions of the oncogenic human papillomaviruses (HPVs) are important in carcinogenesis and immune recognition. We examined the E6 DNA sequence from HPV-16-associated cervical cancers to determine the frequency and degree of variation from the consensus sequence in selected populations. METHODS: Samples positive for HPV-16 were analyzed using polymerase chain reaction followed by automated DNA sequencing: 62 from U.S. women, 20 each from Italian and Indian women, and 21 from Thai women. RESULTS: Of 151 codons, 18 contained 24 base substitutions, reflecting the overall conserved nature of this region. The HPV-16 E6 region from U. S. women showed considerably more sequence variation than that from European and Asian women. Five patterns common to U.S. and European and Asian samples accounted for 78% of all tumor-associated viruses. The E6 regions known to be involved in p53 binding and degradation are involved with a surprising degree of sequence variation, whereas the carboxy end of the molecule is highly conserved. CONCLUSIONS: The area of greatest sequence variation includes a proposed human leukocyte antigen interaction site. A novel large deletion in one sample results in loss of all functional regions of E6. These findings were analyzed for possible significance with regard to immune selection and functional importance of the carboxy end of the E6 protein.