Sera from patients suffering from autoimmune diseases were analyzed for the presence of antibodie... more Sera from patients suffering from autoimmune diseases were analyzed for the presence of antibodies that inhibit adenovirus DNA replication in vitro. DNA replication was studied in a reconstituted system containing purified viral proteins (DNA binding protein, DNA polymerase and the precursor to the terminal protein) and a crude nuclear extract from HeLa cells. About half the autoimmune sera analyzed inhibited DNA replication by more than 50% while only 2 out of 31 control sera showed strong inhibition. The inhibition was caused by the IgG fractions of the sera and was most frequently observed with sera from scleroderma patients. Several lines of evidence indicate that the inhibition is not due to anti-DNA antibodies. The mechanism of inhibition of two strongly inhibitory sera was further investigated. The IgG fractions from these sera blocked DNA chain elongation more than 80% but had no effect on the initiation step or the synthesis of the first 26 nucleotides. Using a dot blot assay and different incubation conditions, evidence was obtained that the inhibition is due to immunorecognition of a nuclear factor from HeLa cells. Two nuclear proteins are known to be required for adenovirus DNA replication, nuclear factors I and II. DNA replication in the presence of purified nuclear factor I instead of a crude nuclear extract was only slightly inhibited by the antisera. In agreement with this, immunorecognition of nuclear factor I could not be detected using a dot blot assay. Since nuclear factor II is not required in our assay system, these results suggest the existence of another nuclear component involved in adenovirus DNA replication which is neutralized by these antibodies.
An alkylhydroxylamine N-acetyltransferase was partially purified from extracts of Kelbsiella pneu... more An alkylhydroxylamine N-acetyltransferase was partially purified from extracts of Kelbsiella pneumoniae. N6-Hydroxylysine, NS-hydroxyornithine and N-methylhydroxylamine were acetylated with decreasing velocity. N6-Acetyl-L-lysine showed noncompetitive inhibition while L-ornithine, Llysine, their a-acetyl derivatives and NS-hydroxylysine were without effect at 0.1 M. A sensitive assay using t4C-labelled acetyl coenzyme A was developed.
An iron-repressible 44-kDa outer membrane protein plays a crucial role in the acquisition of heme... more An iron-repressible 44-kDa outer membrane protein plays a crucial role in the acquisition of heme by the anaerobic bacterium Bacteroides fragilis. The DNA sequence of the gene encoding the 44-kDa protein (hupA) was determined. The hupA gene encodes a protein of 431 amino acid residues with a calculated molecular mass of 48,189 Da. The hupA gene is preceded by an open reading frame of 480 bp that probably encodes a protein with a calculated molecular mass of 18,073 Da. hupA and this open reading frame are likely organized in an operon, and a sequence homologous to the Escherichia coli consensus Fur box was present in the putative promoter region of the operon. Heme-binding studies showed that HupA binds heme. Analysis of the deduced amino acid sequence revealed signature heme-binding consensus motifs, characteristic of heme lyases. Subcellular localization studies in E. coli revealed that HupA was mainly found in the cytoplasmic membrane but not in the outer membrane of E. coli. This suggested that B. fragilis uses another strategy for the translocation of this outer membrane protein across its cell envelope than E. coli does. HupA did not have significant homology with other putative bacterial heme receptors.
Metaplasia in Barrett's esophagus (BE) is characterized by the transition of squamous epithel... more Metaplasia in Barrett's esophagus (BE) is characterized by the transition of squamous epithelium into intestinal-type columnar epithelium. The immune response in BE shares many similarities with the response found in the gut, which is different from the response found in a normal-looking esophagus. Here, we investigated the role of the genes associated with the retinoic acid (RA) pathway in BE, as RA is important not only in shaping the gut's immune response but also in the induction of metaplasia . mRNA was isolated from esophageal and duodenal biopsies from BE ( = 14), reflux esophagitis patients ( = 9), and controls ( = 12). cDNA was made and qPCR was performed. The expression of RALDH1, CYP26A1, MAdCAM1 were similar for both the BE and duodenum, but different when compared to squamous esophageal epithelium. BE was characterized by a higher expression of RALDH2 and FOXP3, compared to the duodenum. In BE, RALDH2 correlated with expression of the myeloid dendritic cell-spec...
Enfermedades Infecciosas y MicrobiologÃa ClÃnica, 2017
The aim of this study was to determine the prevalence of Mycoplasma genitalium infection and the ... more The aim of this study was to determine the prevalence of Mycoplasma genitalium infection and the resistance to macrolides within a general population in Madrid in 2015. We collected 359 urine samples from a general population with symptoms of sexually transmitted infections (STIs). All samples underwent a real-time PCR. For the detection of macrolide resistance, a 283bp fragment of region V of the 23S rRNA gene of M. genitalium was amplified and sequenced. We found a prevalence of 3.34% of M. genitalium and a macrolide resistance rate of 20%. In males, the prevalence was 6.62% and in women 0.96%, being significantly higher in males. The prevalence obtained shows that it is a pathogen to consider in our environment. These findings stress the need for routine testing of M. genitalium infections and would seem to suggest the advisability of resistance testing.
The implementation of polymerase chain reaction (PCR)-based diagnostics of intestinal protozoa ha... more The implementation of polymerase chain reaction (PCR)-based diagnostics of intestinal protozoa has led to higher sensitivity and (subtype) specificity, more convenient sampling, and the possibility for high-throughput screening. PCR for routine detection of human intestinal protozoa in fecal samples is used by an increasing number of clinical laboratories. This paper discusses the recent developments in the diagnosis of intestinal protozoa, with an emphasis on PCR-based diagnostics. Although many reviews have described the technical aspects of PCR-based diagnostics, this review focuses on the clinical consequences that result from the shift from microscopic toward PCR-based diagnostics. Despite its undisputed superiority, the use of PCR comes with challenges that clinicians should be aware of.
The psychrotrophic bacterium Yersinia enterocolitica is characterized by temperature-dependent ad... more The psychrotrophic bacterium Yersinia enterocolitica is characterized by temperature-dependent adaptations. To investigate Y. enterocolitica genes involved in cold adaptation, a mutant restricted in its ability to grow at 5 degrees C was isolated from a transposon mutant library. The transposon insertion site in this psychrotrophy-defective (PD) mutant mapped 16 bp upstream of an open reading frame whose predicted amino acid sequence showed 93% similarity with the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase), encoded by pnp. Expression of this gene was blocked in the PD mutant. However, the introduction of a second copy of pnp, including 0.33 kbp sequences upstream of its coding region, into the chromosome of the PD mutant restored pnp expression as well as the ability to grow at 5 degrees C. Furthermore, the expression of pnp appeared to be temperature dependent: in the parental Y. enterocolitica strain, the levels of both pnp mRNA and PNPase were 1.6-fold higher at 5 degrees C compared with 30 degrees C. A similarly enhanced level of PNPase at 5 degrees C was observed in the merodiploid recombinant strain, which indicates that the 0.33 kbp region upstream of pnp harboured a cold-inducible promoter. A putative cold shock promoter motif (ATTGG) was observed in this region.
cardial vs distal) and tumoral stage. Results: Patients mean age at diagnosis was 69.7 +/-12 year... more cardial vs distal) and tumoral stage. Results: Patients mean age at diagnosis was 69.7 +/-12 years-old. 194 patients (67%) were male. At inclusion, 49 (17%), 27 (9%), 57 (20%) and 157 (54%) patients belonged to stage I-IV, respectively. Curative gastrectomy was performed in 123 (42%) patients. Through follow-up, 196 (67%) patients died: 160 (55%) because of disease progression and the remaining deaths were due to other causes. The fiveyear overall survival rate was 22%. Five-year survival rate was 61%, 42%, 24% and 3%, according to stages I-IV, respectively. TGFB1-869CC carriers had a lower 5-year survival rate than non-carriers (11.4 vs 24.3%, P<0.03 by log-rank test). In the Cox regression analysis, age (
During coevolution with the host, H. pylori has developed unique systems necessary for survival i... more During coevolution with the host, H. pylori has developed unique systems necessary for survival in the human stomach. Concerning competition for nutrients as well as the acidic pH, this niche differs considerably from the environments most other bacteria colonize. ...
RAPD types, Mtz susceptibilities and rdxA sequence-types 'Based on the Wald test from amultivaria... more RAPD types, Mtz susceptibilities and rdxA sequence-types 'Based on the Wald test from amultivariate logistic regression model with year and age as continuous variables infection, presumably from strain 2 to strain I, and indicate that resistance genes are exchanged between different H. pylori strains.
In patients with presumed Barrett esophagus we evaluated clinical risk factors that could predict... more In patients with presumed Barrett esophagus we evaluated clinical risk factors that could predict the presence of intestinal metaplasia and dysplasia in biopsies of columnar-lined esophagus (CLE), independently of histological results. In 908 patients with CLE of length &amp;amp;amp;amp;gt; or = 2 cm, data on age, sex, reflux symptoms, tobacco and alcohol use, medication use, and upper gastrointestinal endoscopy findings were prospectively collected. Multivariate logistic regression analysis was performed, and a model for predicting the histological results was developed. In 127/908 patients, biopsies of CLE did not contain intestinal metaplasia. Of the 781 patients with intestinal metaplasia, 663 patients (85%) had no dysplasia, and 118 (15%) had low grade dysplasia (LGD). The most important predictors for the presence of intestinal metaplasia were length of CLE, size of hiatal hernia, and male sex, while among those with intestinal metaplasia, age and male sex were most important for the presence of LGD. Multivariate combinations of these predictors yielded reliable models, which were able to discriminate intestinal metaplasia well from no intestinal metaplasia (area under receiver operating characteristic [ROC] curve 0.82), but only reasonably discriminated LGD from no dysplasia (area under ROC 0.65). A simple model based on clinical findings can be used to predict the presence of intestinal metaplasia in biopsies from CLE. In contrast, predicting the presence of LGD versus no dysplasia in intestinal metaplasia is more difficult. Predictions from these models may aid decision making on whether a patient with CLE should have surveillance, in view of the known sampling error at endoscopy and interobserver variability at histology.
Sera from patients suffering from autoimmune diseases were analyzed for the presence of antibodie... more Sera from patients suffering from autoimmune diseases were analyzed for the presence of antibodies that inhibit adenovirus DNA replication in vitro. DNA replication was studied in a reconstituted system containing purified viral proteins (DNA binding protein, DNA polymerase and the precursor to the terminal protein) and a crude nuclear extract from HeLa cells. About half the autoimmune sera analyzed inhibited DNA replication by more than 50% while only 2 out of 31 control sera showed strong inhibition. The inhibition was caused by the IgG fractions of the sera and was most frequently observed with sera from scleroderma patients. Several lines of evidence indicate that the inhibition is not due to anti-DNA antibodies. The mechanism of inhibition of two strongly inhibitory sera was further investigated. The IgG fractions from these sera blocked DNA chain elongation more than 80% but had no effect on the initiation step or the synthesis of the first 26 nucleotides. Using a dot blot assay and different incubation conditions, evidence was obtained that the inhibition is due to immunorecognition of a nuclear factor from HeLa cells. Two nuclear proteins are known to be required for adenovirus DNA replication, nuclear factors I and II. DNA replication in the presence of purified nuclear factor I instead of a crude nuclear extract was only slightly inhibited by the antisera. In agreement with this, immunorecognition of nuclear factor I could not be detected using a dot blot assay. Since nuclear factor II is not required in our assay system, these results suggest the existence of another nuclear component involved in adenovirus DNA replication which is neutralized by these antibodies.
An alkylhydroxylamine N-acetyltransferase was partially purified from extracts of Kelbsiella pneu... more An alkylhydroxylamine N-acetyltransferase was partially purified from extracts of Kelbsiella pneumoniae. N6-Hydroxylysine, NS-hydroxyornithine and N-methylhydroxylamine were acetylated with decreasing velocity. N6-Acetyl-L-lysine showed noncompetitive inhibition while L-ornithine, Llysine, their a-acetyl derivatives and NS-hydroxylysine were without effect at 0.1 M. A sensitive assay using t4C-labelled acetyl coenzyme A was developed.
An iron-repressible 44-kDa outer membrane protein plays a crucial role in the acquisition of heme... more An iron-repressible 44-kDa outer membrane protein plays a crucial role in the acquisition of heme by the anaerobic bacterium Bacteroides fragilis. The DNA sequence of the gene encoding the 44-kDa protein (hupA) was determined. The hupA gene encodes a protein of 431 amino acid residues with a calculated molecular mass of 48,189 Da. The hupA gene is preceded by an open reading frame of 480 bp that probably encodes a protein with a calculated molecular mass of 18,073 Da. hupA and this open reading frame are likely organized in an operon, and a sequence homologous to the Escherichia coli consensus Fur box was present in the putative promoter region of the operon. Heme-binding studies showed that HupA binds heme. Analysis of the deduced amino acid sequence revealed signature heme-binding consensus motifs, characteristic of heme lyases. Subcellular localization studies in E. coli revealed that HupA was mainly found in the cytoplasmic membrane but not in the outer membrane of E. coli. This suggested that B. fragilis uses another strategy for the translocation of this outer membrane protein across its cell envelope than E. coli does. HupA did not have significant homology with other putative bacterial heme receptors.
Metaplasia in Barrett's esophagus (BE) is characterized by the transition of squamous epithel... more Metaplasia in Barrett's esophagus (BE) is characterized by the transition of squamous epithelium into intestinal-type columnar epithelium. The immune response in BE shares many similarities with the response found in the gut, which is different from the response found in a normal-looking esophagus. Here, we investigated the role of the genes associated with the retinoic acid (RA) pathway in BE, as RA is important not only in shaping the gut's immune response but also in the induction of metaplasia . mRNA was isolated from esophageal and duodenal biopsies from BE ( = 14), reflux esophagitis patients ( = 9), and controls ( = 12). cDNA was made and qPCR was performed. The expression of RALDH1, CYP26A1, MAdCAM1 were similar for both the BE and duodenum, but different when compared to squamous esophageal epithelium. BE was characterized by a higher expression of RALDH2 and FOXP3, compared to the duodenum. In BE, RALDH2 correlated with expression of the myeloid dendritic cell-spec...
Enfermedades Infecciosas y MicrobiologÃa ClÃnica, 2017
The aim of this study was to determine the prevalence of Mycoplasma genitalium infection and the ... more The aim of this study was to determine the prevalence of Mycoplasma genitalium infection and the resistance to macrolides within a general population in Madrid in 2015. We collected 359 urine samples from a general population with symptoms of sexually transmitted infections (STIs). All samples underwent a real-time PCR. For the detection of macrolide resistance, a 283bp fragment of region V of the 23S rRNA gene of M. genitalium was amplified and sequenced. We found a prevalence of 3.34% of M. genitalium and a macrolide resistance rate of 20%. In males, the prevalence was 6.62% and in women 0.96%, being significantly higher in males. The prevalence obtained shows that it is a pathogen to consider in our environment. These findings stress the need for routine testing of M. genitalium infections and would seem to suggest the advisability of resistance testing.
The implementation of polymerase chain reaction (PCR)-based diagnostics of intestinal protozoa ha... more The implementation of polymerase chain reaction (PCR)-based diagnostics of intestinal protozoa has led to higher sensitivity and (subtype) specificity, more convenient sampling, and the possibility for high-throughput screening. PCR for routine detection of human intestinal protozoa in fecal samples is used by an increasing number of clinical laboratories. This paper discusses the recent developments in the diagnosis of intestinal protozoa, with an emphasis on PCR-based diagnostics. Although many reviews have described the technical aspects of PCR-based diagnostics, this review focuses on the clinical consequences that result from the shift from microscopic toward PCR-based diagnostics. Despite its undisputed superiority, the use of PCR comes with challenges that clinicians should be aware of.
The psychrotrophic bacterium Yersinia enterocolitica is characterized by temperature-dependent ad... more The psychrotrophic bacterium Yersinia enterocolitica is characterized by temperature-dependent adaptations. To investigate Y. enterocolitica genes involved in cold adaptation, a mutant restricted in its ability to grow at 5 degrees C was isolated from a transposon mutant library. The transposon insertion site in this psychrotrophy-defective (PD) mutant mapped 16 bp upstream of an open reading frame whose predicted amino acid sequence showed 93% similarity with the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase), encoded by pnp. Expression of this gene was blocked in the PD mutant. However, the introduction of a second copy of pnp, including 0.33 kbp sequences upstream of its coding region, into the chromosome of the PD mutant restored pnp expression as well as the ability to grow at 5 degrees C. Furthermore, the expression of pnp appeared to be temperature dependent: in the parental Y. enterocolitica strain, the levels of both pnp mRNA and PNPase were 1.6-fold higher at 5 degrees C compared with 30 degrees C. A similarly enhanced level of PNPase at 5 degrees C was observed in the merodiploid recombinant strain, which indicates that the 0.33 kbp region upstream of pnp harboured a cold-inducible promoter. A putative cold shock promoter motif (ATTGG) was observed in this region.
cardial vs distal) and tumoral stage. Results: Patients mean age at diagnosis was 69.7 +/-12 year... more cardial vs distal) and tumoral stage. Results: Patients mean age at diagnosis was 69.7 +/-12 years-old. 194 patients (67%) were male. At inclusion, 49 (17%), 27 (9%), 57 (20%) and 157 (54%) patients belonged to stage I-IV, respectively. Curative gastrectomy was performed in 123 (42%) patients. Through follow-up, 196 (67%) patients died: 160 (55%) because of disease progression and the remaining deaths were due to other causes. The fiveyear overall survival rate was 22%. Five-year survival rate was 61%, 42%, 24% and 3%, according to stages I-IV, respectively. TGFB1-869CC carriers had a lower 5-year survival rate than non-carriers (11.4 vs 24.3%, P<0.03 by log-rank test). In the Cox regression analysis, age (
During coevolution with the host, H. pylori has developed unique systems necessary for survival i... more During coevolution with the host, H. pylori has developed unique systems necessary for survival in the human stomach. Concerning competition for nutrients as well as the acidic pH, this niche differs considerably from the environments most other bacteria colonize. ...
RAPD types, Mtz susceptibilities and rdxA sequence-types 'Based on the Wald test from amultivaria... more RAPD types, Mtz susceptibilities and rdxA sequence-types 'Based on the Wald test from amultivariate logistic regression model with year and age as continuous variables infection, presumably from strain 2 to strain I, and indicate that resistance genes are exchanged between different H. pylori strains.
In patients with presumed Barrett esophagus we evaluated clinical risk factors that could predict... more In patients with presumed Barrett esophagus we evaluated clinical risk factors that could predict the presence of intestinal metaplasia and dysplasia in biopsies of columnar-lined esophagus (CLE), independently of histological results. In 908 patients with CLE of length &amp;amp;amp;amp;gt; or = 2 cm, data on age, sex, reflux symptoms, tobacco and alcohol use, medication use, and upper gastrointestinal endoscopy findings were prospectively collected. Multivariate logistic regression analysis was performed, and a model for predicting the histological results was developed. In 127/908 patients, biopsies of CLE did not contain intestinal metaplasia. Of the 781 patients with intestinal metaplasia, 663 patients (85%) had no dysplasia, and 118 (15%) had low grade dysplasia (LGD). The most important predictors for the presence of intestinal metaplasia were length of CLE, size of hiatal hernia, and male sex, while among those with intestinal metaplasia, age and male sex were most important for the presence of LGD. Multivariate combinations of these predictors yielded reliable models, which were able to discriminate intestinal metaplasia well from no intestinal metaplasia (area under receiver operating characteristic [ROC] curve 0.82), but only reasonably discriminated LGD from no dysplasia (area under ROC 0.65). A simple model based on clinical findings can be used to predict the presence of intestinal metaplasia in biopsies from CLE. In contrast, predicting the presence of LGD versus no dysplasia in intestinal metaplasia is more difficult. Predictions from these models may aid decision making on whether a patient with CLE should have surveillance, in view of the known sampling error at endoscopy and interobserver variability at histology.
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Papers by Hans Kusters