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Biomedical subjects

H Pfister

Publications and source records attributed to H Pfister.

At least 91 records · Page 5Linked to original sources
PubMed1996

Cytokine regulation by virus infection: bovine viral diarrhea virus, a flavivirus, downregulates production of tumor necrosis factor alpha in macrophages in vitro.

Bovine bone marrow-derived macrophages were infected in vitro with noncytopathic or cytopathic strains of bovine viral diarrhea virus. Infection with both biotypes resulted in a decreased production of tumor necrosis factor alpha upon stimulation with heat-inactivated Salmonella dublin or lipopolysaccharide. Other macrophage functions were not downregulated, indicating that the observed effect was not due to a loss in macrophage viability. The downregulated production of tumor necrosis factor alpha in infected macrophages may contribute to the well-documented immunosuppression in animals infected with bovine viral diarrhea virus.

Animals↗
PubMed1996

Molecular diagnosis of persistent human papilloma virus infections.

Persistent infections with human papilloma viruses (HPV) can lead to a wide range of benign and malignant epithelial tumors as cutaneous warts or anogenital cancers. Methods principally available for the laboratory diagnosis of HPV infections are cytology and histology, immunocytochemistry, electron microscopy, serology and methods for the detection of HPV-specific DNA. Among the latter are polymerase chain reaction (PCR), Southern blot, dot blot, filter in situ hybridization, in situ hybridization and direct hybridization assays as Hybrid Capture. The advantages and disadvantages of the different assays are reviewed in this article. HPV DNA testing (and typing) by PCR and Hybrid Capture is clinically useful when combined with cytology in screening programs and for managing patients with equivocal cytology or cervical disease.

Humans↗
PubMed1996

The role of human papillomavirus in anogenital cancer.

More than 30 types of HPV infect the anogenital skin and mucosa, causing condylomas and intraepithelial neoplasia of different severity. On a worldwide basis, HPV 16, 18, and 45 are distinguished by a strong association with high-grade intraepithelial neoplasia and the greatest prevalence in anogenital malignancy. Genetic experiments have assigned oncogenic activity to the viral genes E6, E7, and E5. The encoded proteins interact with and disturb the physiologic functions of cellular proteins that are involved in cell cycle control. The proteins of HPV 16, 18 or related types are most efficient in this regard. Some of these activities lead to genetic instability of the persistently infected human cell. This enhances the probability of mutations in cellular proto-oncogenes and tumor suppressor genes and thus contributes to tumor progression. Mutations in cellular genes devoted to the intracellular surveillance of HPV infections, integration of viral DNA, and deletions or mutations of viral transcription control sequences lead to a significantly increased expression of the E6/E7 genes, which is a consistent characteristic of high-grade intraepithelial neoplasia and cancers. The genetic instability caused by viral oncoproteins and the autocatalytic increase in oncoprotein expression caused by mutations in the viral and cellular genome identify the virus as a major driving force of progression.

Anus Neoplasms↗
PubMed1995

Differentiation-dependent transcription of the epidermodysplasia verruciformis-associated human papillomavirus type 5 in benign lesions.

Human papillomavirus type 5 (HPV 5) induces cutaneous lesions and persists in skin carcinomas of patients with epidermodysplasia verruciformis (EV). We investigated the expression pattern of HPV 5 in biopsies from benign skin lesions of EV patients by cDNA analysis and in situ hybridization. Nine different cDNAs could be generated from total RNA of one of these lesions by reverse transcription and PCR amplification with HPV 5-specific primers. We could identify two major splice donors: one was found in the E6-proximal part of the noncoding region (NCR), and the other just downstream of the first ATG codon of ORF E1. Each of the characterized transcripts was processed at one or the other donor site and the two corresponding leader exons were found in combination with both 3'-early and late exons. Two transcripts appear to be specific for EV-associated papillomaviruses: one species might encode an E1--E2C fusion protein, and the other mRNA (NCR/E2) is probably encoding for the full-length E2 protein. According to the results of the cDNA analysis, riboprobes were designed for in situ hybridization experiments to study the cell differentiation-dependent expression of the different exons. Only the E7/E1 and E4 probes led to strong signals almost throughout the epithelium. The signals generated by the 5'-E2 and E1 probe increased with cell differentiation and were mainly confined to the nucleus. The NCR, E6, E7, L2, and L1 probes yielded more or less strong signals in the terminally differentiated epidermal layers. The difference in the cell differentiation-dependent expression of the 5'-early region exon (probe E7/1) and L2/L1 exons may point to a differentiation-dependent processing of transcripts.

Base Sequence↗
PubMed1995

Experimental Listeria meningoencephalitis. Macrophage inflammatory protein-1 alpha and -2 are produced intrathecally and mediate chemotactic activity in cerebrospinal fluid of infected mice.

In bacterial meningitis, the recruitment of leukocytes across the blood-brain barrier into the central nervous system may be crucial for both elimination of pathogens and tissue injury. In addition to bacterial cell wall products, host factors including chemokines may lead to accumulation of phagocytes within the central nervous system. As shown by Northern analysis, brains of mice infected intracerebrally with Listeria monocytogenes (LM) express mRNA for three chemokines, the macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and MIP-2. The cellular sources of these chemokines comprise both the blood-derived polymorphonuclear leukocytes (PMNs) and monocytes infiltrating the meninges, the ventricular system and the periventricular area. In the course of meningitis a time-dependent increase of MIP-1 alpha and MIP-2 was found in the cerebrospinal fluid (CSF) by ELISA. CSF taken 24 h after infection (CSF-LM24) induced migration of human leukocytes when treated in chemotactic chambers in vitro. Neutralizing Abs to chemokines identified MIP-1 alpha and MIP-2 to be responsible for CSF-LM24 mediated chemotaxis of monocytes and PMNs, respectively. CSF obtained from mock-infected animals contained no MIP-1 alpha or MIP-2 and did not lead to migration of leukocytes. When testing CSF-LM24 on mouse spleen cells, the chemotactic activity detected for mononuclear cells was only partly inhibited by Abs to MIP-1 alpha and -1 beta. Thus, in addition to MIP-1 and -2 other not yet defined chemotactic factors are of importance for recruitment of leukocytes in bacterial meningitis.

Animals↗
PubMed1995

Inducible nitric oxide synthase in cattle. Differential cytokine regulation of nitric oxide synthase in bovine and murine macrophages.

We assessed bovine bone marrow-derived macrophages and monocyte-derived macrophages for expression of inducible nitric oxide synthase (iNOS) activity. Both cell types expressed iNOS activity upon stimulation with Salmonella dublin (S. dublin) or with LPS. A 372-bp fragment of the bovine iNOS mRNA could be amplified by reverse transcription-PCR from mRNA of stimulated macrophages. Cloning and sequencing of the fragment revealed a high degree of homology to human hepatocyte, rat vascular smooth muscle cell, and mouse macrophage iNOS both at the nucleotide (87 to 92%) and amino acid levels (94 to 97%). iNOS mRNA was expressed maximally 6 h after stimulation with S. dublin, whereas maximal nitrite accumulation in supernatants was measured at 24 to 48 h. Significant differences with regard to cytokine regulation of iNOS were observed between murine and bovine macrophages cultured under identical conditions. The most striking difference was the inability of homologous IFN-gamma to induce iNOS both at the level of nitrite production and of mRNA expression in bovine macrophages. TNF-alpha, IL-2, and IL-1 alone or together with IFN-gamma neither induced iNOS nor primed bovine macrophages for enhanced iNOS expression or activity upon stimulation with S. dublin. RhuIL-4, but not rhuTGF-beta, down-regulated S. dublin-induced iNOS activity and mRNA expression in bovine macrophages. Thus, an enzyme with a high degree of homology to rodent iNOS is inducible by stimulation of bovine macrophages with bacteria, but induction and regulation by cytokines occur under more restricted conditions than in rodent macrophages.

Amino Acid Oxidoreductases↗
PubMed1995

Increased concentrations of transferrin in the urine and serum of cattle with cardiomyopathy.

Bovine cardiomyopathy affects adult cattle of the Simmental/red Holstein and Holstein-Friesian breeds and is characterised clinically by signs of congestive heart failure. Animals with cardiomyopathy suffered a marked renal loss of transferrin (Tf). The urinary concentration of Tf discriminated very well between healthy and affected cattle, 93 per cent of the affected and 97 per cent of the healthy cows being identified correctly. In spite of the severe liver congestion and renal loss of Tf, cattle with cardiomyopathy had considerably higher concentrations of serum Tf than healthy cattle. This increase may help to compensate for the low serum concentration of iron in the affected animals. The pronounced changes in the concentrations of iron and transferrin in the serum had little effect on the haemogram of the diseased animals.

Animals↗
PubMed1995

Variation of human papillomavirus type 6 (HPV-6) and HPV-11 genomes sampled throughout the world.

We examined the genomic diversity of human papillomavirus type 6 (HPV-6) and HPV-11 isolates from different parts of the world by comparing the nucleotide sequences of part of the long control region of three reference clones and 62 HPV-6 and 40 HPV-11 isolates from Africa, Europe, Asia, and North and South America. The genomic sequence of the HPV-6b reference type had to be amended by inclusion of a 94-bp segment, which is also present with minor differences in HPV-6a. Aside from two small inserts typical of all variants related to HPV-6a and three inserts found in HPV-11 variants, no major alterations to the size of the long control regions of these viruses were observed. This corrects the previous impression that these two HPV types are highly polymorphic. Altogether, 19 HPV-6 and 10 HPV-11 variant genomes could be distinguished, and most of the differences were due to point substitutions. The variants of either type were continuously connected in phylogenetic trees rather than clustered separately into subtype groups. Thirteen mutations, namely, the two HPV-6a inserts and 11 substitutions in HPV-6 or HPV-11 variants, reduced the dissimilarity between the types, but they bridged only a small fraction of the genetic distance between the two types. Genomes more obviously intermediate between HPV-6 and HPV-11 were not found and probably do not exist any more. A single HPV-11 variant was found in Africa, but otherwise, no significant correlations of HPV-6 or HPV-11 variants with geography or ethnicity of the patient cohort were found. Functional analysis of diverse enhancer variants showed activities that differed two-to threefold, and it must be considered that transcriptional differences may alter the biology or pathology of these viruses. Similar variants were found in lesions from anatomically different sites and in both benign and malignant lesions.

Base Sequence↗
PubMed1995

Human papillomavirus DNA in genital cancers, metastases, and lymph nodes.

To evaluate the utility of human papillomavirus (HPV) DNA as molecular marker for diagnostic screening of lymph nodes for cancer metastases, we examined 16 involved and 124 histologically cancer-free nodes of 34 patients with cervical, vulval, or vaginal carcinomas associated with HPV 6, 16, 18, 31, 33, 45, or X. All metastases of HPV-positive tumors contained viral DNA of the same type as the primary cancer but in one case the copy number of viral genomes was dramatically reduced. In a varying proportion of histologically normal nodes of 7 of 23 patients with HPV 16-positive cancers HPV 16 DNA was revealed by Southern blot hybridization and/or PCR. Histologic reevaluation showed a metastasis in one case. Three examples of tumor heterogeneity regarding physical state or copy number of viral DNA are presented. Tumor heterogeneity is discussed as possible explanation for apparently inconsistent results such as HPV-negative metastases of HPV-positive tumors.

Base Sequence↗
PubMed1994

Prevalence of deletions of YY1-binding sites in episomal HPV 16 DNA from cervical cancers.

Expression of the oncogenes E6 and E7 of human papillomavirus 16 (HPV 16) appears enhanced in pre-malignant and malignant genital tumors. We recently identified a transcriptional silencer upstream of the oncogene promoter P97, comprising 4 binding sites for the cellular YY1 protein. The analysis of the long transcriptional control regions (LCR) of episomal HPV 16 DNAs from primary tumors and lymph-node metastases of 6 patients with cervical cancer revealed deletions and point mutations of YY1 binding sites in 4 cases. To test for the activity of the P97 promoter, the mutated LCRs were cloned in a luciferase reporter gene vector. A point mutation in YY1-recognition site 4, which prevents DNA-protein interaction, did not affect promoter activity, probably due to compensation by the overlapping YY1-binding site 3. However, 5.5- to 6.5-fold increased luciferase expression was obtained under the control of 3 shortened LCRs lacking 2 to 4 YY1-binding sites. A point mutation in YY1-recognition site 2, which was previously shown to stimulate P97 3.5-fold, could be detected in the HPV 16 LCRs from both primary tumor and metastasis, indicating that the mutation is a stable characteristic of HPV 16 DNA associated with the individual cancer. These findings suggest that deletions or mutations of YY1-binding sites play a significant role in over-expression of viral oncogenes and tumor progression.

Base Sequence↗
PubMed1994

The E6/E7 promoter of extrachromosomal HPV16 DNA in cervical cancers escapes from cellular repression by mutation of target sequences for YY1.

Human papillomavirus type 16 (HPV16) induces squamous intraepithelial lesions of the cervical mucosa which may develop into invasive cancer. The expression of viral oncogenes in advanced neoplasias appears increased relative to the proliferating cell layers of low grade lesions raising questions about molecular mechanisms of deregulation of transcription. In a lymph node metastasis of a cervical cancer, we observed full-length HPV16 plasmids and molecules with a small deletion, which was mapped to the long control region (LCR). Both wild type and shortened LCR were amplified by PCR, cloned into the promoter test plasmid pBLCAT6 and sequenced to identify a 107 bp deletion from position 7794 to 7901 in the short LCR. CAT expression in cervical cancer-derived HT3, SiHa and CaSki cells appeared 5- to 6-fold increased under the control of the short LCR. This could be traced back to elevated levels of mRNA initiated at the viral oncogene promoter. A slight further increase in CAT expression was noted in the presence of the HPV16 E2 protein which is probably due to the deletion of one E2 binding site and consequent relief from E2 repression. Computer-assisted sequence analysis and band-shift experiments with purified YY1 protein and wild type or mutated oligonucleotides identified four binding sites for this cellular transcriptional repressor within the promoter-proximal segment of the HPV16 LCR, three of which were removed by the deletion. A LCR fragment comprising these YY1 binding sites was cloned in front of the heterologous thymidine kinase gene promoter and suppressed CAT expression 3- to 4-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1994

Change in sensitivity to lipopolysaccharide during the differentiation of human monocytes to macrophages in vitro.

Mononuclear phagocytes in distinct differentiation stages and cultured under different conditions were tested for their sensitivity towards lipopolysaccharide (LPS), using procoagulant activity (PCA) expression and tumor necrosis factor (TNF) production as indices. The response of mature monocyte-derived macrophages differed from that of freshly isolated monocytes 1) by higher levels of constitutive PCA, 2) by responding to approximately 1,000-fold lower concentrations of LPS with PCA and TNF production, and 3) by a faster rise in PCA and TNF production. Due to the high constitutive level of PCA expression, the PCA stimulation index for LPS was low in macrophages when compared with that in monocytes. Thus, during differentiation to macrophages, human monocytes acquire increased sensitivity to LPS (2 orders of magnitude more sensitive than a sensitive turbidimetric Limulus amoebocyte lysate assay). This exquisite sensitivity to LPS is expressed regardless of whether LPS is offered in the presence or absence of lipopolysaccharide binding protein-containing serum. This points to as yet uncharacterized pathways of high affinity interaction between LPS and macrophages.

Blood Coagulation Factors↗
PubMed1994

Intrathecal synthesis of major immunoglobulin classes in inflammatory diseases of the canine CNS.

The IgG index, IgM and IgA contents in cerebrospinal fluid and serum were examined retrospectively using an enzyme-linked immunosorbent assay (ELISA) in 69 dogs with inflammatory central nervous system (CNS) diseases of various aetiologies. Fifteen normal dogs were used as controls. After measuring IgG and albumin contents in serum and cerebrospinal fluid (CSF), the IgG index was calculated according to the formula of Link and Tibbling to demonstrate intrathecal immunoglobulin synthesis. A surprisingly high number of animals with encephalitis, including dogs with protracted diseases such as chronic distemper encephalitis and granulomatous meningoencephalomyelitis, showed in addition to an elevated IgG production evidence of intrathecal IgM and IgA production. The highest values of intrathecal as well as systemic IgA levels were found in dogs suffering from steroid responsive meningitis-arteritis. It was concluded that the control of the humoral immune response in the brain differs from that in other tissues. Because of striking similarities between dogs and humans in respect to humoral neuroimmunological reactions, the dog can be considered to be a useful animal model for the study of the intrathecal humoral immune response.

Animals↗
PubMed1994

Blood serum transferrin concentration in cattle in various physiological states, in veal calves fed different amounts of iron, and in cattle affected by infectious and non-infectious diseases.

Transferrin (Tf) concentrations were determined in cattle in various physiological states, in energy-deficient (ketotic) cows, in situations of several acute and chronic infections, after endotoxin administration and in animals with bovine leucocyte adhesion deficiency (BLAD). Tf concentrations varied between 1.5 and 8.5 g/l and in healthy animals were in the range of 2.0 and 6.6 g/l. Tf concentrations in adult animals were smaller than in young animals and increased in veal calves with iron deficiency above 8 g/l, resulting in a negative correlation between Hb and Tf. In veal calves total iron binding capacity (TIBC) and Tf concentration were rather closely correlated (r = 0.63). Chronic infectious diseases (such as paratuberculosis) were characterized by relatively low Tf levels (below 2 g/l), while during acute infections, after endotoxin-administration and during ketosis Tf concentrations were not changed.

Animals↗
PubMed1994

Low-affinity E2-binding site mediates downmodulation of E2 transactivation of the human papillomavirus type 8 late promoter.

The constitutively active promoter P7535 of the epidermodysplasia verruciformis-associated human papillomavirus type 8 (HPV8) is transactivated by the viral E2 protein. The distribution of potential E2-binding sites (ACCN6GGT) in the viral transcription control region is highly conserved among epidermodysplasia verruciformis-associated human papillomaviruses and differs completely from that of other papillomaviruses. To investigate the role of E2-binding sites P0 to P4 in P7535 regulation, we analyzed their binding affinities in gel retardation experiments using a full-length HPV8 E2 protein expressed from a recombinant baculovirus. Binding site P1 within a transcriptional silencer showed the highest affinity, followed by P0 within the L1 gene and P3 downstream of P7535. P2, 33 nucleotides upstream of the mRNA cap site, and P4 were very weak binders. There is some indication that the number of A/T pairs in the nonconserved core of the recognition sequence is critical for the binding of HPV8 E2. Transient transfection experiments were carried out with an HPV8 E2 expression vector and reporter plasmids containing mutated E2-binding sites in the context of the HPV8 regulatory region. The knockout of the strongest binding site P1 sufficed to clearly diminish transactivation. P0, P3, and P4 mutations had little effect on their own, whereas double mutations P01 and P34 strongly reduced E2 inducibility. Both mutations in P2 severely affected constitutive promoter activity but had opposite effects on transactivation. They revealed an inverse correlation between E2-binding strength and the extent of E2 transactivation. This finding suggests that P2 mediates a negative control of P7535 by E2, counteracting E2 transactivation exerted via the four distal E2 target sequences.

Base Sequence↗
PubMed1994

Transcriptional silencer of the human papillomavirus type 8 late promoter interacts alternatively with the viral trans activator E2 or with a cellular factor.

The noncoding region of the highly oncogenic, epidermodysplasia verruciformis-associated human papillomavirus type 8 contains a negative regulatory element (NRE). Quantitative RNase protection analysis confirmed that the NRE sequence acts as a silencer of transcription. A 38-bp sequence upstream of late promoter P7535 down-regulated expression from the homologous P7535 promoter, as well as the heterologous tk gene promoter, independently of its orientation relative to the test promoters. It also reduced gene expression when cloned downstream of the transcription units. Transient expression assays with keratinocytes and fibroblasts of epidermodysplasia verruciformis patients and controls demonstrated that the NRE activity is not cell specific. Gel retardation tests suggested that NRE specifically interacts with only one nuclear factor. Mutational analysis identified three NRE mutants which no longer formed a detectable DNA-protein complex but still repressed transcription, indicating that protein-DNA interaction is not relevant for the silencer function. The NRE contains a binding site of viral trans activator protein E2. It was shown that expression of E2 overrides the inhibitory effect of the NRE sequences. Binding of E2 and that of the cellular factor were mutually exclusive. The bifunctional nature of NRE acting as a silencer and a target site for viral trans activator E2 offers an interesting opportunity to regulate the switch from early to late transcription in the human papillomavirus life cycle.

Base Sequence↗