P. J. M. Roholl

Host cell membrane proteins on human immunodeficiency virus type 1 after in vitro infection of H9 cells and blood mononuclear cells. An immuno-electron microscopic study.

Human immunodeficiency virus type 1 (HIV-1)-infected H9 and blood mononuclear cells (MNCs) were studied by immunogold electron microscopy for the presence of HIV-1 gag p24 protein, env gp41 and gp120 proteins, and host cell molecules CD4, CD11a, CD25, CD54, CD63, HLA class I and HLA-DR. Uninfected H9 cells and MNC membranes labelled for CD4, HLA class I and class II, and, at low density, CD11a and CD54; lysosomal structures in the cytoplasm labelled for CD63. The infected cell surface showed immunolabelling for HIV-1 proteins, as did budding particle-like structures. Immunogold labelling of the cell membrane for CD4 was almost non-existent. The level of immunolabelling for CD11a and CD54 on infected cells was greater than that on uninfected cells; this is presumably related to a state of activation during virus synthesis. Budding particle-like structures and free virions in the intercellular space were immunogold-labelled for all host cell markers investigated. This was confirmed by double immunogold labelling using combinations of HIV-1 gag p24 labelling and labelling for the respective host cell molecule. We conclude that virions generated in HIV-1-infected cells concentrate host-derived molecules on their envelope. Also molecules with a prime function in cellular adhesion concentrate on the virion.

A study to analyze the origin of tumor cells in malignant fibrous histiocytomas a multiparametric characterization

To study the derivation of tumor cells of malignant fibrous histiocytomas (MFH), their phenotypical marker profile was investigated and compared with those of malignant histiocytosis (MH) and of different types of soft tissue tumors (STT). The presence of the following markers was investigated: on paraffin sections, alpha‐1‐antichymotrypsin (ACT); on frozen sections antigens associated with lymphocytes, macrophages and fibroblasts, the enzymes acid phosphatase, nonspecific esterase, and beta‐glucuronidase; and, on isolated and cultured cells, the receptors for EA‐gamma and complement. Furthermore, the capacity to phagocytose sensitized erythrocytes and carbon particles was studied in vitro. MFH tumor cells and a part of other types of STT shared the expression of ACT and lysosomal enzymes with MH. They differed, however, from MH by the absence of monocyte/macrophage‐associated antigens and by the expression of fibroblast‐associated antigens, which property they had in common with other STT. MFH tumor cells were not able to form rosettes or to phagocytose Ig‐sensitized erythrocytes, but they showed phagocytosis of carbon particles. The results strongly indicate that MFH tumor cells originate from (primitive) fixed mesenchymal cells and are not related to monocyte‐derived histiocytes.

The relevance of the DNA index and proliferation rate in the grading of benign and malignant soft tissue tumors

The relevance of the DNA index (DI) to malignancy grading and the relationships between mitotic score, Ki‐67 score, and the proportion of cells in the G1‐phase (G1PF) of the cell cycle, as proliferative indicators, were investigated in benign (n = 8) and malignant (n = 46) soft tissue sarcomas. Although DI was not found to be related to the histology of the sarcomas, it was associated with malignancy grade, i.e., 14%, 25%, 42%, and 86% of benign, Grade 1, Grade 2, and Grade 3, respectively had a DI>1. The relevance of aneuploidy in benign tumors is discussed and the literature reviewed. A subgroup of the malignant tumors (n = 13) had both a low mitotic score and a low Ki‐67 value. However, a distinct correlation between Ki‐67 and the mitotic score could not be shown. For malignant tumours G1PF was related to DI, i.e., low G1PF occurred in tumors with diploid DNA content. Low Ki‐67 scores were observed in 59% of diploid tumors and in 20% of aneuploid tumors. However, it appeared that G1PF and Ki‐67 were not correlated. In conclusion, (1) benign and Grade 1 tumors were predominantly diploid with high G1PF and low Ki‐67 values, (2) most of Grade 3 tumors were aneuploid (86%) with low G1PF and high Ki‐67 values, and (3) the group of Grade 2 tumors could be divided into two subgroups either with the characteristics displayed by benign and Grade 1 tumors (DI = 1, low Ki‐67 scores, and high G1PF) or with the characteristics exhibited by Grade 3 tumors (DI>1, high Ki‐67 scores, and low G1PF). Hence, supplementary to the grading of soft tissue tumors, DI, G1PF, and Ki‐67 score could be useful as prognostic parameters in soft tissue tumors.

Distribution of Chlamydia pneumoniae in the Human Arterial System and Its Relation to the Local Amount of Atherosclerosis Within the Individual

BackgroundChlamydia pneumoniae has been suggested to play a role in the origin of atherosclerosis. We studied the prevalence of C pneumoniae at multiple locations in the arterial system within the same individual. Studying the association between atherosclerosis and C pneumoniae within the individual excludes confounding by interindividual variability. Methods and ResultsPostmortem, the presence in the intima/plaque and media of C pneumoniae membrane protein was determined by use of a C pneumoniae-specific monoclonal antibody. In 24 individuals, 33 arterial locations were studied (n=738 segments). Area stenosis was determined in adjacent cross sections. In all individuals, immunostaining of C pneumoniae was observed in ≥1 artery. The highest prevalences were observed in the abdominal aorta (67%), internal and common iliac arteries (41%), and coronary arteries (33%). The lowest prevalences were observed in the radial (0%) and cerebral (2%) arteries. Within the individual, area stenosis was larger in cross sections with immunoreactivity compared with cross sections without immunoreactivity (31.0±11.9% versus 14.3±6.1%, respectively;P <0.001). In the individual, immunoreactivity was observed in 15±10% of the arteries (range, 3% to 45%). Between individuals, the percentage of arteries with immunoreactivity to C pneumoniae was associated with the average area stenosis throughout the arterial system (r2=0.56, P <0.001). ConclusionsC pneumoniae was mostly observed at locations that are related to clinically relevant features. Within the individual, the distribution of C pneumoniae is associated with the distribution of atherosclerosis. The role of the microorganism in atherosclerotic disease remains to be elucidated.

Expression of insulin-like growth factors (IGFs), their receptors and IGF binding protein-3 in normal, benign and malignant smooth muscle tissues

To assess the role of insulin-like growth factors (IGFs) in growth and transformation of normal (myometrium) and tumorous smooth muscle cell (SMC) tissues, in situ hybridization (ISH) analysis for insulin-like growth factor I and II (IGF-I and IGF-II) mRNAs was combined with detection of IGF peptides, their receptors and IGF binding protein-3 (IGFBP-3). mRNAs for both IGFs were detected in smooth muscle cells in normal, benign and malignant SMC tissues, together with the IGF peptides, both IGF receptors and IGFBP-3. This suggests an autocrine role for both IGFs. Leiomyomas had higher IGF-I peptide levels and higher levels of type I IGF receptors than myometrium, supporting the idea that IGFs play a role in the growth and transformation of these tumours. Low-grade leiomyosarcomas contained more IGF-II mRNAs than myometrium and leiomyoma, fewer type II IGF/mannose 6-phosphate receptors and less IGFBP-3 than myometrium and, in addition, fewer IGF-I mRNAs and type I IGF receptors than leiomyoma. Intermediate- and high-grade leiomyosarcomas had intermediate levels of IGF-II mRNAs and peptide, ranging between those in myometrium and low-grade leiomyosarcomas. Thus, growth and transformation of leiomyosarcomas may be regulated by IGF-II, although more markedly in low-grade than in high-grade leiomyosarcomas. In conclusion, the various categories of SMC tissues are associated with a distinct expression pattern of the IGF system. This suggests that each category of SMC tumours arises as a distinct entity and that there is no progression of transformation in these tissues.

A highly attenuated recombinant human respiratory syncytial virus lacking the G protein induces long-lasting protection in cotton rats

BackgroundRespiratory syncytial virus (RSV) is a primary cause of serious lower respiratory tract illness for which there is still no safe and effective vaccine available. Using reverse genetics, recombinant (r)RSV and an rRSV lacking the G gene (ΔG) were constructed based on a clinical RSV isolate (strain 98-25147-X).ResultsGrowth of both recombinant viruses was equivalent to that of wild type virus in Vero cells, but was reduced in human epithelial cells like Hep-2. Replication in cotton rat lungs could not be detected for ΔG, while rRSV was 100-fold attenuated compared to wild type virus. Upon single dose intranasal administration in cotton rats, both recombinant viruses developed high levels of neutralizing antibodies and conferred comparable long-lasting protection against RSV challenge; protection against replication in the lungs lasted at least 147 days and protection against pulmonary inflammation lasted at least 75 days.ConclusionCollectively, the data indicate that a single dose immunization with the highly attenuated ΔG as well as the attenuated rRSV conferred long term protection in the cotton rat against subsequent RSV challenge, without inducing vaccine enhanced pathology. Since ΔG is not likely to revert to a less attenuated phenotype, we plan to evaluate this deletion mutant further and to investigate its potential as a vaccine candidate against RSV infection.

Autoreactivity against induced or upregulated abundant self-peptides in HLA-A*0201 following measles virus infection

Infectious agents have been implied as causative environmental factors in the development of autoimmunity. However, the exact nature of their involvement remains unknown. We describe a possible mechanism for the activation of autoreactive T cells induced by measles virus (MV) infection. The display of HLA-A*0201 associated peptides obtained from MV infected cells was compared with that from uninfected cells by mass spectrometry. We identified two abundant self peptides, IFI-6-16(74-82) and Hsp90beta(570-578), that were induced or upregulated, respectively, following infection. Their parental proteins, the type I interferon inducible protein IFI-6-16, and the beta chain of heat shock protein 90, have not been involved in MV pathogenesis. MV infection caused minor and major changes in the intracellular expression patterns of these proteins, possibly leading to altered peptide processing. CD8+ T cells capable of recognizing the self-peptides in the context of HLA-A*0201 were detectable at low basal levels in the neonatal and adult human T cell repertoire, but were functionally silent. In contrast, peptide-specific producing IFN-gamma producing effector cells were present in MV patients during acute infection. Thus, MV infection induces an enhanced display of self-peptides in MHC class I, which may lead to the temporary activation of autoreactive T cells.

In vivo antibody response and in vitro CTL activation induced by selected measles vaccine candidates, prepared with purified Quil A components.

Semipurified Quil A and purified Quil A were used to prepare well-characterized subunit vaccine candidates against measles. Variation in the relative amounts of the measles virus (MV) fusion (F) protein, Quil A-components and lipids did not influence induction of antibody responses in mice, but had a pronounced effect on the capacity to induce cytotoxic T cell (CTL) activity of a CD8(+) MV F-protein specific human T cell clone in vitro. A characteristic MV iscom preparation based on the combined use of HPLC-purified Quil A-components QA-3 and QA-22 (QA-3/22) efficiently induced CTL activity in vitro. Comparable results were obtained by mixing beta-propiolactone inactivated MV with iscom-matrix QA-3/22 or free QA-22. On the basis of the data presented it was concluded that these three preparations are interesting MV vaccine candidates for further evaluation in pre-clinical experiments in a primate model.

Determination of the growth fraction in monoclonal gammopathy with the monoclonal antibody Ki-67

The monoclonal antibody Ki‐67 reacts with a nuclear antigen that is present only in proliferating cells. The proportion of Ki‐67 positive cells may therefore serve as a reliable measurement for the growth fraction in normal and neoplasmic cell populations. We have tested the significance of the MoAb Ki‐67 in the classification of monoclonal gammopathy and compared the results with the plasma cell labelling index. In benign monoclonal gammopathy the percentage of Ki‐67 positive plasma cells (median 1.6%) was significantly lower than in untreated multiple myeloma (median 9.6). Among the patients with more than 10% Ki‐67 positive plasma cells there were some very short survivors.

Distribution of actin isoforms in sarcomas: An immunohistochemical study

The actin immunophenotype of eight benign mesenchymal tumors, 14 nonsarcomatoid tumors, and 46 sarcomatoid tumors was studied, using monoclonal antibodies (MoAb) specific for alpha-smooth muscle actin (clone 1A4), alpha- and gamma-smooth muscle actin (designated CGA7), and muscle actin (designated HHF35) on frozen sections. Tumor cells of nonsarcomatoid tissues were not reactive, but all leiomyomas and five of the seven leiomyosarcomas reacted with the three MoAbs. One leiomyosarcoma was immunoreactive for the MoAb 1A4 only. One of the six malignant schwannomas showed staining for muscle actin (HHF35). The 22 malignant fibrous histocytomas (MFH) expressed these actin isoforms in various degrees. One case immunoreacted with all three MoAbs, three reacted with 1A4 only, seven reacted with CGA7 and HHF35, and two reacted with HHF35 only. Nine MFHs were not immunoreactive for any of the MoAbs specific for (smooth) muscle and actin. In addition, the expression of desmin and collagen type IV was investigated for the group of leiomyosarcomas and MFHs. Desmin was found in five leiomyosarcomas and in two MFHs. Collagen type IV was seen in all leiomyosarcomas, and was seen weakly in a few small areas in four MFHs. When we take into account the expression of all markers tested [( smooth] muscle actin, desmin, and collagen type IV), then six of the 22 MFHs were unreactive for all these markers. Five of these six tumors were located intramuscularly, whereas only half of the total number of MFH cases had an intramuscular location. The fact that 15 of 22 MFHs displayed one or more markers linked with (smooth) muscle differentiation suggests that some of the MFHs may be classified as poorly differentiated leiomyosarcomas, and that MFH is not a unique entity.