MiR expression profiles of paired primary colorectal cancer and metastases by next-generation sequencing.

MicroRNAs (miRs) have been recognized as promising biomarkers. It is unknown to what extent tumor-derived miRs are differentially expressed between primary colorectal cancers (pCRCs) and metastatic lesions, and to what extent the expression profiles of tumor tissue differ from the surrounding normal tissue. Next-generation sequencing (NGS) of 220 fresh-frozen samples, including paired primary and metastatic tumor tissue and non-tumorous tissue from 38 patients, revealed expression of 2245 known unique mature miRs and 515 novel candidate miRs. Unsupervised clustering of miR expression profiles of pCRC tissue with paired metastases did not separate the two entities, whereas unsupervised clustering of miR expression profiles of pCRC with normal colorectal mucosa demonstrated complete separation of the tumor samples from their paired normal mucosa. Two hundred and twenty-two miRs differentiated both pCRC and metastases from normal tissue samples (false discovery rate (FDR) <0.05). The highest expressed tumor-specific miRs were miR-21 and miR-92a, both previously described to be involved in CRC with potential as circulating biomarker for early detection. Only eight miRs, 0.5% of the analysed miR transcriptome, were differentially expressed between pCRC and the corresponding metastases (FDR <0.1), consisting of five known miRs (miR-320b, miR-320d, miR-3117, miR-1246 and miR-663b) and three novel candidate miRs (chr 1-2552-5p, chr 8-20656-5p and chr 10-25333-3p). These results indicate that previously unrecognized candidate miRs expressed in advanced CRC were identified using NGS. In addition, miR expression profiles of pCRC and metastatic lesions are highly comparable and may be of similar predictive value for prognosis or response to treatment in patients with advanced CRC.

Small bowel adenocarcinoma copy number profiles are more closely related to colorectal than to gastric cancers.

BACKGROUND: Small bowel adenocarcinoma (SBA) is a rare cancer and consequently, the options for clinical trials are limited. As they are treated according to either a colorectal or a gastric cancer regimen and the molecular biology of a tumor is a pivotal determinant for therapy response, chromosomal copy number aberrations were compared with the colorectal and gastric adenocarcinomas. MATERIALS AND METHODS: A total of 85 microsatellite stable (MSS) adenocarcinomas from the stomach, colorectum and small bowel were selected from existing array comparative genomic hybridization (aCGH) datasets. We compared the aCGH profiles of the three tumor sites by supervised analysis and hierarchical clustering. RESULTS: Hierarchical clustering revealed substantial overlap of 27 SBA copy number profiles with matched colorectal adenocarcinomas but less overlap with profiles of gastric adenocarcinomas. DNA copy number aberrations located at chromosomes 1p36.3-p34.3, 4p15.3-q35.2, 9p24.3-p11.1, 13q13.2-q31.3 and 17p13.3-p13.2 were the strongest features discriminating SBAs and colorectal adenocarcinomas from gastric adenocarcinomas. CONCLUSIONS: We show that MSS SBAs are more similar to colorectal than to gastric cancer, based on the 27 genome-wide DNA copy number profiles that are currently available. These molecular similarities provide added support for treatment of MSS small bowel cancers according to colorectal cancer regimens.

Integrative analysis of array-comparative genomic hybridisation and matched gene expression profiling data reveals novel genes with prognostic significance in oesophageal adenocarcinoma.

BACKGROUND AND AIMS: The incidence of oesophageal adenocarcinoma (OAC) has been increasing rapidly with a dismal survival rate of less than 20%. Understanding the genomic aberrations and biology of this cancer may enhance disease interventions. This study aimed to use genome-wide genomic and expression data to enhance the understanding of OAC pathogenesis and identify groups with differential outcomes. METHODS: Array-comparative genomic hybridisation (aCGH) analysis was carried out on 56 fresh frozen OAC resection samples with long-term clinical follow-up data. Samples with aberrations were further analysed with whole-genome single-nucleotide polymorphism arrays to confirm aCGH findings. Matched gene expression microarray data were used to identify genes with high copy number-expression correlations. Nested-multiplex PCR on DNA from microdissected specimens and fluorescence in situ hybridisation assays were used for target validation. Immunohistochemistry on the same cohort and independent samples (n=371) was used for subsequent validation. Kaplan-Meier survival analyses were performed based on aCGH data after unsupervised K-means clustering (K=5, 50 iterations) and immunohistochemistry data. RESULTS: aCGH identified 17 common regions (>5% samples) of gains and 11 common regions of losses, including novel regions in OAC (loci 11p13 and 21q21.2). Integration of aCGH data with matched gene expression microarray data highlighted genes with high copy number-expression correlations: two deletions (p16/CDKN2A, MBNL1) and four gains (EGFR, WT1, NEIL2, MTMR9). Immunohistochemistry demonstrated protein over-expression of targets with gains: EGFR (10%), WT1 (20%), NEIL2 (14%) and MTMR9 (25%). These targets individually (p<0.060) and in combination had prognostic significance (p=0.008). On the genomic level, K-means clustering identified a cluster (32% of cohort) with differential log(2) ratios of 16 CGH probes (p<4×10(-7)) and a worse prognosis (median survival=1.37 years; p=0.015). CONCLUSIONS: Integration of aCGH and gene expression data identified copy number aberrations and novel genes with prognostic potential in OAC.

DNA copy number profiles of primary tumors as predictors of response to chemotherapy in advanced colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is biologically a heterogeneous disease, which may affect response to drug therapy. We investigated the correlation of genome-wide DNA copy number profiles of primary tumors with response to systemic chemotherapy in advanced CRC. PATIENTS AND METHODS: DNA was isolated from formaldehyde-fixed paraffin-embedded primary tumors of 32 patients with advanced CRC, which were selected based on either a good response (n = 16) or a poor response (n = 16) to first-line combination therapy with capecitabine and irinotecan. High-resolution DNA copy number profiles were obtained by means of 30 K oligonucleotide-based array comparative genomic hybridization (aCGH). RESULTS: Unsupervised hierarchical cluster analysis of the aCGH data revealed two clusters of 19 and 13 tumors, respectively, and cluster membership showed a significant correlation with response status (P < 0.03). The nonresponders had fewer chromosomal alterations compared with the responders, in particular less losses were found (P < 0.03). Most prominent differences between the two groups were losses of regions 18p11.32-q11.2 (P < 0.02) and 18q12.1-q23 (P < 0.03), which were more frequently observed in responders. CONCLUSIONS: Differences in DNA copy number profiles of primary CRCs are associated with response to systemic combination chemotherapy with capecitabine and irinotecan. Responders overall had more chromosomal alterations, especially loss of chromosome 18.

The genomic profile of human malignant glioma is altered early in primary cell culture and preserved in spheroids.

Screening of therapeutics relies on representative cancer models. The representation of human glioblastoma by in vitro cell culture models is questionable. We obtained genomic profiles by array comparative genomic hybridization of both short- and long-term primary cell and spheroid cultures, derived from seven glioblastomas and one anaplastic oligodendroglioma. Chromosomal copy numbers were compared between cell cultures and spheroids and related to the parental gliomas using unsupervised hierarchical clustering and correlation coefficient. In seven out of eight short-term cell cultures, the genomic profiles clustered further apart from their parental tumors than spheroid cultures. In four out of eight samples, the genetic changes in cell culture were substantial. The average correlation coefficient between parental tumors and spheroid profiles was 0.89 (range: 0.79-0.97), whereas that between parental tumors and cell cultures was 0.62 (range: 0.10-0.96). In two out of three long-term cell cultures progressive genetic changes had developed, whereas the spheroid cultures were genetically stable. It is concluded that genomic profiles of primary cell cultures from glioblastoma are frequently deviant from parental tumor profiles, whereas spheroids are genetically more representative of the glioblastoma. This implies that glioma cell culture data have to be handled with the highest caution.

Cross-platform array comparative genomic hybridization meta-analysis separates hematopoietic and mesenchymal from epithelial tumors.

A series of studies have been published that evaluate the chromosomal copy number changes of different tumor classes using array comparative genomic hybridization (array CGH); however, the chromosomal aberrations that distinguish the different tumor classes have not been fully characterized. Therefore, we performed a meta-analysis of different array CGH data sets in an attempt to classify samples tested across different platforms. As opposed to RNA expression, a common reference is used in dual channel CGH arrays: normal human DNA, theoretically facilitating cross-platform analysis. To this aim, cell line and primary cancer data sets from three different dual channel array CGH platforms obtained by four different institutes were integrated. The cell line data were used to develop preprocessing methods, which performed noise reduction and transformed samples into a common format. The transformed array CGH profiles allowed perfect clustering by cell line, but importantly not by platform or institute. The same preprocessing procedures used for the cell line data were applied to data from 373 primary tumors profiled by array CGH, including controls. Results indicated that there is no apparent feature related to the institute or platform and that array CGH allows for unambiguous cross-platform meta-analysis. Major clusters with common tissue origin were identified. Interestingly, tumors of hematopoietic and mesenchymal origins cluster separately from tumors of epithelial origin. Therefore, it can be concluded that chromosomal aberrations of tumors from hematopoietic and mesenchymal origin versus tumors of epithelial origin are distinct, and these differences can be picked up by meta-analysis of array CGH data. This suggests the possibility of prospectively using combined analysis of diverse copy number data sets for cancer subtype classification.

Gastric cancers in young and elderly patients show different genomic profiles.

Although most gastric cancers occur in elderly patients, a substantial number of cases of this common disease occur in young patients. Gastric cancer is a heterogeneous disease at the genomic level and different patterns of DNA copy number alterations are associated with different clinical behaviour. The aim of the present study was to explore differences in DNA copy number alterations in relation to age of onset of gastric cancer. DNA isolated from 46 paraffin-embedded gastric cancer tissue samples from 17 patients less than 50 years of age [median 43 (21-49) years] and 29 patients greater than or equal to 70 years of age [median 75 (70-83) years] was analysed by genome-wide microarray comparative genomic hybridization (array CGH) using an array of 5000 BAC clones. Patterns of DNA copy number aberrations were analysed by hierarchical cluster analysis of the mode-normalized and smoothed log(2) ratios of tumour to normal reference fluorescence signal intensities using TMEV software, after which cluster membership was correlated with age group. In addition, supervised analysis was performed using CGH Multi-array. Hierarchical cluster analysis of the array CGH data revealed three clusters with different genomic profiles that correlated significantly with age (p = 0.006). Cluster 1 mainly contained young patients, while elderly patients were divided over clusters 2 and 3. Chromosome regions 11q23.3 and 19p13.3 contributed most to age-related differences in tumour profiles. Gastric cancers of young and old patients belong to groups with different genomic profiles, which likely reflect different pathogenic mechanisms of the disease.

Profiling of apoptosis genes allows for clinical stratification of primary nodal diffuse large B-cell lymphomas.

Intrinsic resistance of lymphoma cells to apoptosis is a probable mechanism causing chemotherapy resistance and eventual fatal outcome in patients with diffuse large B cell lymphomas (DLBCL). We investigated whether microarray expression profiling of apoptosis related genes predicts clinical outcome in 46 patients with primary nodal DLBCL. Unsupervised cluster analysis using genes involved in apoptosis (n = 246) resulted in three separate DLBCL groups partly overlapping with germinal centre B-lymphocytes versus activated B-cells like phenotype. One group with poor clinical outcome was characterised by high expression levels of pro-and anti-apoptotic genes involved in the intrinsic apoptosis pathway. A second group, also with poor clinical outcome, was characterised by high levels of apoptosis inducing cytotoxic effector genes, possibly reflecting a cellular cytotoxic immune response. The third group showing a favourable outcome was characterised by low expression levels of genes characteristic for both other groups. Our results suggest that chemotherapy refractory DLBCL are characterised either by an intense cellular cytotoxic immune response or by constitutive activation of the intrinsic mediated apoptosis pathway with concomitant downstream inhibition of this apoptosis pathway. Consequently, strategies neutralising the function of apoptosis-inhibiting proteins might be effective as alternative treatment modality in part of chemotherapy refractory DLBCL.

Micro-array analysis of resistance for gemcitabine results in increased expression of ribonucleotide reductase subunits.

To study in detail the relation between gene expression and resistance against gemcitabine, a cell line was isolated from a tumor for which gemcitabine resistance was induced in vivo. Similar to the in vivo tumor, resistance in this cell line, C 26-G, was not related to deficiency of deoxycytidine kinase (dCK). Micro-array analysis showed increased expression of ribonucleotide reductase (RR) subunits M1 and M2 as confirmed by real time PCR analysis (28- and 2.7-fold, respectively). In cell culture, moderate cross-resistance (about 2-fold) was observed to 1-ss-D-arabinofuranosylcytosine (ara-C), 2-chloro-2'deoxyadenosine (CdA), LY231514 (ALIMTA), and cisplatin (CDDP), and pronounced cross-resistance (>23-fold) to 2',2'-difluorodeoxyuridine (dFdU) and 2',2'-difluorodeoxyguanosine (dFdG). Culture in the absence of gemcitabine reduced resistance as well as RRM1 RNA expression, demonstrating a direct relationship of RRM1 RNA expression with acquired resistance to gemcitabine.

Normal IncA expression and fusogenicity of inclusions in Chlamydia trachomatis isolates with the incA I47T mutation.

To investigate the correlation between the incA I47T mutation in Chlamydia trachomatis and the nonfusogenic phenotype, the incA genes of 25 isolates were sequenced. Four major sequence types were identified. Seven isolates (28%) had the I47T mutation. Isolates representing the four sequence types expressed IncA in the membrane of one large single inclusion. In conclusion, the incA I47T mutation is not associated with the nonfusogenic phenotype.

PCR assessment of Chlamydia trachomatis infection of semen specimens processed for artificial insemination.

In order to ascertain the microbiological quality of stored semen specimens processed for artificial insemination by a donor (AID), we developed a PCR assay targeting the chlamydial plasmid to detect Chlamydia trachomatis in semen. The lower limit of detection of this assay corresponded to 2.5 to 5 elementary bodies per microl of semen. A total of 669 cryopreserved ejaculates from 97 asymptomatic donors were tested for C. trachomatis infection. Twelve ejaculates, originating from four donors, were found to be positive, indicating a 4% prevalence of C. trachomatis infection among the donor population studied. Cross-contamination between the cryopreserved specimens in the storage container was studied by typing using sequence analysis of PCR-amplified omp1 genes of the strains. Two donors were infected with serovar E, one was infected with serovar F, and one was infected with serovar K. For two donors, the duration of C. trachomatis positivity could be assessed. One donor donated C. trachomatis-positive semen for at least 4 successive months, and the other did so for at least 16 months. The occurrence of C. trachomatis infection in cryopreserved donor semen indicates that ejaculates from donors not tested for a C. trachomatis infection just prior to donation should be tested for infection by a direct test such as the PCR described here. Direct testing of semen specimens will detect not only donors with an active infection but also C. trachomatis-infected ejaculates already stored and will thus improve the microbiological quality of AID, since discrepancies in the presence of C. trachomatis in urine and semen specimens have been reported.

Stimulation of the adherence of Haemophilus influenzae to human lung epithelial cells by antimicrobial neutrophil defensins.

Patients with chronic obstructive pulmonary disease (COPD) frequently have recurrent lower respiratory tract infections with nonencapsulated Haemophilus influenzae. The infected mucosa of these patients is infiltrated with neutrophils, which upon activation may release antimicrobial peptides, including defensins. It was shown that defensins isolated from neutrophils or from sputum samples of COPD patients did not kill H. influenzae from these patients, but they did stimulate its adherence to human bronchial epithelial cells in a time- and dose-dependent manner. Maximal stimulation was observed after 3 h in the presence of > or = 10 micrograms/mL defensins, resulting in 65 +/- 36 cfu/cell (61-fold increase). The enhanced adherence was not solely due to charge effects and was specifically blocked by alpha 1-proteinase inhibitor. Because adherence is the first step in the onset of respiratory tract infections, our findings indicate that neutrophil defensins likely contribute to the pathogenesis of H. influenzae infection in the lower respiratory tract.

The fimbria gene cluster of nonencapsulated Haemophilus influenzae.

The occurrence of fimbria gene clusters in nonencapsulated Haemophilus influenzae strains from chronic bronchitis patients (n = 58), patients with acute otitis media (n = 13), and healthy carriers (n = 12) was determined by DNA hybridization and PCR, based on sequences of fimbriate H. influenzae type b. Although an average of 18% of all nonencapsulated strains had a fimbria gene cluster consisting of hifA to hifE inserted in the chromosome between purE and pepN, differences in the frequency of fimbria cluster-positive strains were observed, depending on the source of isolates. The compositions of the fimbria gene clusters of seven strains from chronic bronchitis patients and one strain from an otitis media patient were analyzed in more detail. After enrichment for fimbria expression, the promoter of the gene cluster contained 10 TA repeats (n = 2), leading to optimal positioning between the -10 and -35 promoter regions. The promoter regions of five fimbria-negative strains were sequenced; four were found to have nine TA repeats, and one had only four TA repeats. The protein sequence of three ganglioside GM1-specific HifA adhesins consisted of conserved regions intermingled with regions of sequence diversity. hifA appeared to be flanked by intergenic regions that varied between strains and contained both direct and inverted DNA repeats. Since noncoding DNA between hifA and purE has not been found in H. influenzae type b, these DNA sequences are probably not essential for fimbria expression. An analysis of strains lacking the gene cluster revealed the presence of similar sequences in 13 of 15 strains from chronic bronchitis patients, 5 of 5 strains from otitis media patients, and 3 of 5 strains from healthy carriers. The lengths of these intergenic regions were the same for multiple isolates of strains obtained during persistent infections. The presence or absence and the composition of the fimbria gene cluster and other sequences between the flanking genes purE and pepN suggest that the fimbria gene cluster was originally contained on a mobile element.

Molecular variation in the major outer membrane protein P5 gene of nonencapsulated Haemophilus influenzae during chronic infections.

During the course of persistent infections by nonencapsulated Haemophilus influenzae in patients with chronic bronchitis, the major outer membrane protein (MOMP) P5 varies in molecular weight. The nature of this variability was determined by DNA sequence analysis of the P5 gene from five different H. influenzae strains and their seven MOMP P5 variants which were isolated from patients with chronic infections of the lower respiratory tract. Analysis of the P5 sequence data from the different strains revealed four well-defined, heterogeneous regions. These regions of variable sequence appeared to correspond to the regions of the gene encoding the putative surface-exposed loops of MOMP P5. The MOMP P5 variants with alterations in MOMP P5 were shown to result from DNA point mutations and codon deletions. In addition, in three variants derived sequentially from one H. influenzae strain, a frameshift mutation resulted in the formation of a stop codon in the region encoding the signal sequence of the MOMP P5 gene. Strikingly, all nucleotide substitutions in the MOMP P5 loop regions of variants were nonsynonymous, suggesting that variants with alterated amino acid compositions of the surface-exposed parts of MOMP P5 obtained a selective advantage during persistence of the infection by nonencapsulated H. influenzae in chronic bronchitis patients.

Haemophilus influenzae infections in patients with chronic obstructive pulmonary disease despite specific antibodies in serum and sputum.

The titer and specificity of antibodies to the infecting Haemophilus influenzae was determined in sera and sputa from 27 patients with chronic obstructive pulmonary disease (COPD) to analyze the specific immune response. COPD patients had significantly higher serum IgG and IgA antibody titers than 13 healthy controls (mean IgG titers 12,302 and 5,623, respectively; mean IgA titers, 2,398 and 912; p less than 0.001). The mean IgM titers were comparable: 501 and 447, respectively. Specific IgA antibodies were also detectable in the sputum of the COPD patients (mean IgA antibody titer, 776). The local antibody production was determined by calculating the relative coefficient of excretion (RCE) to albumin. The mean RCE of 89.1 for IgA indicated statistically significant local production (p less than 0.02), in contrast to a nonsignificant increase for IgG (mean RCE of 3.6). Specific IgM was below the detection level. Immunoblotting experiments showed that the antibodies in sera from COPD patients and controls were directed against most of the outer membrane proteins of H. influenzae, with individual differences between IgG, IgA, and IgM. The IgA and IgG antibodies in serum had a similar specificity as those in sputum. The appearance or persistence of H. influenzae coincided with minor changes in antibody titer and specificity. From these results we conclude that COPD patients are infected with H. influenzae despite the presence of at least as many antibodies in sputum and serum as in controls and that these antibodies are directed against a variety of antigenic determinants of the infecting strain.

Endogenous and exogenous reinfections by Haemophilus influenzae in patients with chronic obstructive pulmonary disease: the effect of antibiotic treatment on persistence.

To analyze whether exacerbations in chronic obstructive pulmonary disease (COPD) coincide with reinfection by Haemophilus influenzae, 16 COPD patients were studied longitudinally for 3 years. Exacerbations coincided with reinfection by H. influenzae, either endogenous, by a strain with a DNA fingerprint indistinguishable from the strain previously present but with another major outer membrane protein (MOMP) pattern (2 patients), or exogenous, by a strain with a different DNA fingerprint and MOMP pattern (3 patients). The other patients, remaining in an infectious state without clear exacerbations for longer periods, were persistently infected by a particular H. influenzae strain (median persistence time, 5.5 months; range, 2-23 months). Of 8 antibiotic-treated patients, 7 remained infected by H. influenzae with the same DNA fingerprint, although all strains were sensitive to the antibiotics prescribed. Results of the study suggested that exacerbations in COPD patients coincide with endogenous or exogenous reinfection by H. influenzae, persistently infected patients keep the same H. influenzae strain for longer periods, and antibiotic treatment was not effective in eradicating H. influenzae.

Antigenic drift of Haemophilus influenzae in patients with chronic obstructive pulmonary disease.

Differences in the major outer membrane protein b,c (molecular weight, 39,000 to 41,000) of related Haemophilus influenzae strains isolated from the sputum of patients with chronic obstructive pulmonary disease were analyzed biochemically and immunologically. Protein b,c was isolated from a total of six related H. influenzae strains from two chronic obstructive pulmonary disease patients. After CNBr digestion of the proteins, the differences in size appeared in the larger of the two fragments. Trypsin and chymotrypsin digests of proteins from related H. influenzae strains showed that proteins differed by only a few peptides or were very similar, in contrast to the peptide maps of proteins from nonrelated strains. Peptide analysis of b,c proteins from related H. influenzae strains by high-performance liquid chromatography after Staphylococcus aureus V8 protease digestion and amino acid analysis of corresponding fractions revealed highly comparable patterns, indicating only minor differences in the amino acid sequences of these proteins. Immunization of rabbits with intact bacteria of four related H. influenzae strains resulted in a strong anti-protein b,c antibody response in all animals. In three of four rabbits, antibodies specific for the b,c protein of the strain used for immunization were elicited, indicating that the changed proteins contained specific immunodominant epitopes. Anti-protein b,c antibodies promoted strain-specific, complement-dependent, bactericidal activity. From these results, we conclude that H. influenzae shows antigenic drift in immunodominant epitopes, caused by small changes in amino acid composition of the b,c protein. Antibodies to these epitopes promote complement-dependent bactericidal activity.

Nonculture detection of Haemophilus influenzae in sputum with monoclonal antibodies specific for outer membrane lipoprotein P6.

Isolation of Haemophilus influenzae from sputum is hampered by overgrowth by other microorganisms or by antibiotic treatment of the patient. To overcome this problem in the detection of H. influenzae, an in situ immunoperoxidase staining technique was developed with monoclonal antibody (MAb) 8BD9, immunoglobulin subclass G2a. MAb 8BD9 appeared to be directed to an epitope on the outer membrane lipoprotein P6 of H. influenzae. The species specificity of MAb 8BD9 was analyzed by staining isolates from different bacterial species. MAb 8BD9 reacted with all 300 H. influenzae strains tested and with H. aegyptius and H. haemolyticus. Twenty-six of 30 H. parainfluenzae strains, other Haemophilus species, and other bacterial species often isolated from sputum were not stained. The staining technique was compared with culture of 845 routinely obtained sputum samples from patients with respiratory tract infections. In 829 sputa (98.1%), the results of both techniques were in agreement; 173 were positive for H. influenzae, and 656 were negative. With 14 sputum samples, the staining method gave a positive result for H. influenzae, but the bacterium was not cultured. This could be ascribed to antibiotic treatment of the patient (n = 7), the presence of other MAb 8BD9-positive Haemophilus species in the sputum (n = 5), and overgrowth by swarming Proteus mirabilis or by Branhamella catarrhalis. In the immunoperoxidase- and Gram-stained smears of two sputum samples, no bacteria were seen, although some H. influenzae was cultured. On the basis of these results, we conclude that immunoperoxidase staining with MAb 8BD9 is a fast and reliable extension of the available detection techniques for H. influenzae.

Comparison of two nonculture techniques for detection of Hemophilus influenzae in sputum. In situ hybridization and immunoperoxidase staining with monoclonal antibodies.

Two nonculture methods, in situ hybridization and immunoperoxidase staining with monoclonal antibodies, were compared for the detection of Hemophilus influenzae in 184 sputa. For in situ hybridization, a biotin-labeled probe of total genomic DNA of H influenzae type b was prepared that hybridizes specifically with H influenzae, H parainfluenzae, H hemolyticus, and H parahemolyticus DNA. Immunoperoxidase staining was done with monoclonal antibody 8BD9 directed against outer membrane protein P6 of H influenzae. Both techniques detected Hemophilus in sputum equally well and were superior to culture: all 30 sputum samples culture-positive for H influenzae were positive on both nonculture tests, and 13 additional positive sputum samples were detected from which Hemophilus was not cultured. The higher sensitivity of the nonculture tests was mainly attributed to culture failure because of overgrowth of H influenzae by other bacteria, especially in patients with cystic fibrosis. The immunoperoxidase staining technique appeared slightly easier and quicker to perform than the in situ hybridization test. For the in situ DNA hybridization probe, DNA can be prepared from any strain of H influenzae. The immunoperoxidase test requires monoclonal antibody 8BD9 but has a higher specificity than the hybridization technique. Both techniques can be reliably applied, especially for the detection of Hemophilus in sputum of patients with cystic fibrosis.

Changes in outer membrane proteins of nontypable Haemophilus influenzae in patients with chronic obstructive pulmonary disease.

Five individual colonies of Haemophilus influenzae were isolated from each of one to three cultures of sputum collected from 18 patients with chronic obstructive pulmonary disease (COPD). The isolates were studied to investigate whether the major outer membrane proteins (MOMPs) changed during persistence. The relationship between isolates was analyzed by fingerprinting their chromosomal DNA. The fingerprints of eight strains (isolated from eight patients) with various MOMP compositions were different, whereas fingerprints of isolates with identical MOMP compositions were indistinguishable. In 12 patients, two or more strains with different MOMP compositions were found; one strain was isolated from the sputum samples of each of the six remaining patients. In seven of the 12 patients, strains with different MOMPs but with indistinguishable fingerprints were found. The differences were found in proteins b,c (five patients) and d (five patients). In patients with COPD, the MOMPs of H. influenzae are subject to changes that may enable this bacterium to escape immunological defense mechanisms.