Diagnostic precision of image-guided multisampling core needle biopsy of suspected lymphomas in a primary care hospital.

We evaluated the diagnostic quality of image-guided multisampling core needle biopsy (CNB) in patients investigated for suspected lymphoma in a primary care hospital. A total of 112 patients were consecutively assessed during a 3-year period. There were 80 lymphoid site biopsies and 32 non-lymphoid site biopsies. Eight to nine cores were obtained from different parts of the biopsy site. Two cores were systematically frozen, allowing for further morphological, immunochemistry and molecular studies. The diagnostic yield of CNB for malignancy was 100%. Only 47% (41/87) of patients with initial suspicion of lymphoma were finally diagnosed with Lymphoma. The diagnostic yield of CNB for lymphoma typing was 98% (62/63), according to the WHO classification. The diagnostic yield of CNB for complete lymphoma subtyping/grading was 86% (54/63). The diagnostic yield of CNB for a definite diagnosis of benignity was only 47% (8/17). In a primary care setting, multisampling CNB is a minimally invasive, and very accurate procedure for confirming malignancy in patients with suspected lymphoma, presenting with superficial/deep-seated, lymphoid/non-lymphoid site targets. With a very high diagnostic yield for lymphoma typing and a high diagnostic yield for complete lymphoma subtyping/grading a therapeutic decision can be taken in most patients.

Evaluation of tumour vascularisation in two rat sarcoma models for studying isolated lung perfusion. Injection route determines the origin of tumour vessels.

Isolated cytostatic lung perfusion (ILP) is an attractive technique allowing delivery of a high-dose of cytostatic agents to the lungs while limiting systemic toxicity. In developing a rat model of ILP, we have analysed the effect of the route of tumour cell injection on the source of tumour vessels. Pulmonary sarcomas were established by injecting a sarcoma cell suspension either by the intravenous (i.v.) route or directly into the lung parenchyma. Ink perfusion through either pulmonary artery (PA) or bronchial arteries (BA) was performed and the characteristics of the tumour deposits defined. i.v. and direct injection methods induced pulmonary sarcoma nodules, with similar histological features. The intraparenchymal injection of tumour cells resulted in more reliable and reproducible tumour growth and was associated with a longer survival of the animals. i.v. injected tumours developed a PA-derived vascular tree whereas directly injected tumours developed a BA-derived vasculature.

Sequence analysis and in vitro expression of genes 6 and 11 of an ovine group B rotavirus isolate, KB63: evidence for a non-defective, C-terminally truncated NSP1 and a phosphorylated NSP5.

An ovine group B rotavirus (GBR) isolate, KB63, was isolated from faeces of a young goat with diarrhoea in Xinjiang, People's Republic of China. Sequence determination and comparison of genes 6 and 11 with the corresponding sequences of GBR strains ADRV and IDIR showed that they were the cognate genes encoding NSP1 and NSP5, respectively. While the overall identities of nucleotide sequences between these two genes and the corresponding genes of strains ADRV and IDIR were in the range 52.6-57.2%, the identities of deduced amino acid sequences were only 34.9-46.3%. These results demonstrate that the substantial diversity of NSP1 observed among group A rotaviruses (GAR) also exists within GBRs and that a high degree of diversity also exists among NSP5 of GBRs, in contrast to GAR NSP5. The NSP1 gene of KB63 contains three ORFs, whereas the NSP1 genes of other GBR strains contain only two. ORFs 2 and 3 of the KB63 gene may be derived from a single ORF corresponding to ORF2 of other GBR strains by the usage of a stop codon created by an upstream single base deletion and single point mutations. In vitro expression studies showed that ORFs 1 and 2, but not 3, of gene 6 can be translated, suggesting that ORF2 may encode a C-terminally truncated, potentially functional product. It may play a role, together with the product of ORF1, in virus replication, as the virus can be passaged further in kids. Similarly, gene 11 can be translated in vitro. Like its counterpart in GARs, the protein encoded by gene 11 was shown to be phosphorylated in vitro.

The development of an antigen capture polymerase chain reaction assay to detect and type human enteroviruses.

Antigen capture polymerase chain reaction (AC-PCR) is a technique that combines the advantages of PCR with those of antibody mediated methods, to detect and type human enteroviruses. Virus particles are captured by specific antisera and RNA is released by heat denaturation to generate the substrate for reverse transcription and PCR. Use of this technique results in purification of human enteroviruses from tissue culture and 10% faecal samples in a serotype-specific manner allowing both rapid detection and a direct correlation between serological and genetic typing methods. The sensitivity of AC-PCR was comparable with that of PCR protocols employing a conventional organic solvent based extraction procedure.

Risks for transmission of hepatitis C virus during artificial insemination.

OBJECTIVE: To identify risks of hepatitis C virus transmission by semen from infected donors. DESIGN: Case report. SETTING: Assisted fertility clinic. PATIENTS: Hepatitis C virus-infected semen donor and recipients of his donations. INTERVENTION: Testing for hepatitis C virus by serology and polymerase chain reaction. MAIN OUTCOME MEASURES: Detection of hepatitis C virus antibodies and viral RNA. RESULTS: Hepatitis C virus RNA was detected in the semen donation before but not after purification; none of the recipients of the donors samples were found to have antibodies to hepatitis C virus. CONCLUSIONS: Hepatitis C virus RNA can be detected in semen donations from infected donors; purification of donations before insemination significantly reduces the amount of viral RNA in the semen pellet.

Molecular epidemiology of hepatitis C virus infection amongst intravenous drug users in rural communities.

The prevalence of hepatitis C virus (HCV) infection amongst a group of intravenous drug users (IVDUs) resident in West Suffolk (East Anglia, England) was investigated and compared with the prevalence of infection with hepatitis B virus (HBV) and human immunodeficiency virus (HIV). In addition, both the level of HCV persistence, as defined by detection of viral RNA, and the HCV genotypes present in this population were determined. It was found that HCV antibodies were present in 59% of those tested; by comparison 22% had antibodies to HBV and 1% antibodies to HIV. HCV RNA was found in 44% of those with HCV antibody. HCV genotype 1 was the most prevalent within this population although both genotypes 2 and 3 were also represented.

Internal quality assurance in a clinical virology laboratory. II. Internal quality control.

AIMS: In April 1991 additional quality control procedures were introduced into the virology section of the Clinical Microbiology and Public Health Laboratory, Cambridge. Internal quality control (IQC) samples were gradually included in the serological assays performed in the laboratory and supplemented kit controls and standard sera. METHODS: From April 1991 to December 1993, 2421 IQC procedures were carried out with reference sera. RESULTS: The IQC samples were evaluated according to the Westgard rules. Violations were recorded in 60 of 1808 (3.3%) controls and were highest in the IQC samples of complement fixation tests (25/312 (8%) of controls submitted for complement fixation tests). CONCLUSIONS: The inclusion of IQC samples in the serological assays performed in the laboratory has highlighted batch to batch variation in commercial assays. The setting of acceptable limits for the IQC samples has increased confidence in the validity of assay results.

Internal quality assurance in a clinical virology laboratory. I. Internal quality assessment.

AIMS: In April 1991 an internal quality assessment scheme (IQAS) was introduced into the virology section of the Clinical Microbiology and Public Health Laboratory, Cambridge. The IQAS was established to identify recurring technical and procedural problems, to check the adequacy of current techniques, and to calculate the frequency of errors. METHODS: Between April 1991 and December 1993, 715 anonymous clinical serum samples were submitted to the laboratory to test 3245 individual procedures of diagnostic viral serology. RESULTS: A total of 485 (14.9%) procedural and 61 (1.9%) technical discrepancies were observed, the technical discrepancies mainly being recorded in complement fixation tests. Twenty two (0.7% of total procedures) of the technical discrepancies were diagnostically significant. CONCLUSIONS: Evaluation criteria developed with the introduction of IQAS to viral serology, and technical and procedural discrepancies are assessed. As yet, IQAS has not been introduced to other sections of the diagnostic virology laboratory (virus isolation, electron microscopy, immunofluorescence, and enzyme linked immunosorbent assays for viral and chlamydial antigens).

Use of recombinant vectors derived from herpes simplex virus 1 mutant tsK for short-term expression of transgenes encoding cytoplasmic and membrane anchored proteins in postmitotic polarized cortical neurons and glial cells in vitro.

We constructed three recombinant vectors derived from the herpes simplex virus type 1 mutant tsK, each of which contained a different transgene under the control of the herpes simplex virus type 1 immediate early 3 promoter inserted into the thymidine kinase locus: the prokaryotic enzymes beta-galactosidase and chloramphenicol acetyl transferase, and a fusion gene consisting of human tissue inhibitor of metalloproteinases linked to the last exon of Thy-1, which encodes for a glycosyl-phosphatidyl-inositol membrane anchor. Infection of postmitotic neocortical and hippocampal neurons in low-density primary cultures with these vectors, achieved reliable expression of all three foreign gene products in various neocortical cell types, e.g. pyramidal neurons, non-pyramidal neurons, and glial cells. The percentage of neurons expressing transgenes ranged from 1 to 46% depending on the multiplicity of infection (highest assayed = 5); the percentage of glial cells expressing transgenes ranged from 0.5 to 98% (highest multiplicity assayed = 3.4). Expression of transgenes could be detected for up to three days in approximately 20% of neurons infected at a multiplicity of infection of 1. Infection of neurons with tk K-derived recombinant vectors inhibited their protein synthesis by 40-50% at a multiplicity of infection of 10, but no effect was observed at a multiplicity of infection of 1. Infection of glial cells with the same vectors at a multiplicity of infection of 1 inhibited protein synthesis by more than 90%. Analysis of neuronal viability at different times post-infection indicated that more than 98% of neurons expressing transgenes 48 h post-infection were viable. Thus, low-density neuronal cultures can be used to assess the efficiency of herpes simplex virus type 1-derived gene transfer vectors and transgene expression in developing cortical postmitotic cells, before and after they establish polarity. In addition, we show that two cytoplasmic enzymes, beta-galactosidase and chloramphenicol acetyl transferase, are able to diffuse freely in the cytoplasm reaching even growth cones in young neurons, while the chimeric protein tissue inhibitor of metalloproteinases/Thy-1 is correctly targeted to the plasma membrane via a glycosyl-phosphatidylinositol anchor. This model system should be useful for investigation of cellular and molecular aspects of the development and establishment of neuronal polarity, as well as for analysis of signals involved in protein targeting in postmitotic neurons.

A "string-of-beads" vaccine, comprising linked minigenes, confers protection from lethal-dose virus challenge.

We have previously demonstrated that induction of antiviral cytotoxic T lymphocytes (CTL), in the absence of antiviral antibodies, can confer protection against a lethal-dose virus challenge. Here we extend those findings as follows. First, three discrete viral CTL epitopes expressed from minigenes encoding peptides as short as 12 amino acids can be recognized when expressed from recombinant vaccinia virus; second, concentrating on two of the three epitopes, we show that these vaccinia virus recombinants can confer protection in a major histocompatibility complex (MHC)-restricted manner; third, the minigenes can be fused to generate a "string of beads," and the close proximity of the two epitopes within one oligopeptide does not disrupt recognition of either epitope; fourth, this string-of-beads vaccine, in contrast to the single epitope vaccines, can protect on both MHC backgrounds; and, fifth, CTL to different epitopes may act synergistically, as protection is improved when the vaccine contains more than one CTL epitope for a given MHC background.

Novel LCMV-specific H-2k restricted CTL clones recognize internal viral gene products and cause CNS disease.

H-2k (C3H/Hej) cytotoxic T lymphocytes (CTL) specific for lymphocytic choriomeningitis virus (LCMV) were cloned. Three clones recognizing internal viral antigens were studied. One such CTL clone recognized neither the glycoprotein nor nucleoprotein encoded by the viral short RNA segment, but reacted with a protein encoded by the long RNA segment, either the viral polymerase, or the Z protein. This one clone, in addition to primary CTL harvested from immunized C3H mice, failed to lyse target cells expressing the Z protein, suggesting recognition was to the viral polymerase. Two other clones recognized the viral nucleoprotein, amino acids 93-100, as determined by protein deletion and peptide mapping studies. When introduced directly into the central nervous systems of LCMV-infected histocompatible mice, all clones were active in vivo and capable of causing immunopathologically mediated death.

Identification of two protein binding sites within the varicella-zoster virus major immediate early gene promoter.

Binding sites for cellular proteins in the promoter of the varicella-zoster virus (VZV) major immediate early (IE) gene were investigated. Protein binding was detected at sequence motifs possessing homology to the CCAAT element and an ATF/AP-1-like binding site, and recognition of the ATF/AP-1 site was apparently facilitated by occupation of the CCAAT site. Gene expression directed by the VZV major IE promoter was stimulated by the adenovirus 5, 289 amino acid EIA gene product. The implications of the results for VZV gene expression and replication are discussed.

The product of varicella-zoster virus gene 62 autoregulates its own promoter.

Varicella-zoster (VZV) gene 62 encodes a protein with a predicted Mr of 140,000 (140K) which has considerable amino acid identity with the major immediate early (IE) protein Vmw175 (ICP4) of herpes simplex virus type I (HSV-1). Vmw175 is an essential virus polypeptide with a pivotal role in the activation of early and late viral gene expression and also in the repression of IE gene expression. The VZV 140K protein has been shown to function as a strong transcriptional activator in transfection assays and largely complements for the loss of Vmw175 function in HSV-1. We report the results of cotransfection experiments which demonstrate that the 140K protein strongly represses expression from its own promoter, that of gene 62, thus establishing further functional similarity between it and Vmw175. However, whereas Vmw175 can substitute for the 140K protein in repression of the gene 62 promoter, the 140K protein does not repress the HSV-1 IE3 promoter in the reciprocal experiment. The integrity of a domain of Vmw175 (designated region 2), previously shown to be crucial for repression of the HSV-1 IE3 promoter, is also required for repression of the gene 62 promoter. Moreover, a similar requirement for the highly similar region 2 of the 140K protein for repression is demonstrated, suggesting that VZV 140K protein and HSV-1 Vmw175 autoregulate IE gene expression by a related mechanism.

Control of expression of the varicella-zoster virus major immediate early gene.

The cis-acting DNA sequences and trans-acting proteins that control the expression of the major immediate early (IE) gene of varicella-zoster virus (VZV) were investigated. The location of the IE mRNA 5' terminus was determined by primer extension and S1 nuclease analyses and the functional activities of DNA sequences upstream of this site were analysed by a transfection assay. The VZV IE promoter exhibited low activity in BHK and HeLa cells, but was transactivated by the herpes simplex virus type 1 (HSV-1) virion protein Vmw65. DNA sequences between positions -131 and +57 were responsible for promoter activity, whereas sequences between -410 and -131 mediated the response to Vmw65. Two short elements in the -410 to -131 region formed protein-DNA complexes with HeLa cell nuclear proteins and formed a ternary complex when Vmw65 was added. One of the elements, ATGTAAATGAAAT, possessed a strong similarity to the HSV-1 TAATGARAT. The VZV homologue of Vmw65, encoded by open reading frame (ORF) 10, failed to trans-activate expression from HSV-1 or VZV IE promoters and did not form a ternary complex with functional TAATGARAT elements and HeLa cell proteins. Therefore, stimulation of VZV IE transcription by Vmw65 can occur by a mechanism similar to that employed by HSV-1, but VZV ORF 10 does not function as a trans-activator of IE gene expression.

Construction and characterization of a herpes simplex virus type 1 mutant unable to transinduce immediate-early gene expression.

A herpes simplex virus mutant, in1814, possessing a 12-base-pair insertion in the gene encoding the transinducing factor Vmw65 has been constructed. The insertion abolished the ability of Vmw65 to transinduce immediate-early (IE) gene expression and to form a protein-DNA complex with cell proteins and the IE-specific regulatory element TAATGAGAT. Accumulation of IE RNA 1 and 2 was reduced four- to fivefold in in1814-infected cells, but the level of IE RNA 4 was reduced only by twofold, and IE RNA 3 was unaffected. Mutant in1814 had a high particle/PFU ratio, but many of the particles, although unable to form plaques, were capable of normal participation in the early stages of infection at high multiplicity of infection. The defect of in1814 was overcome partially by transfection of a plasmid encoding the IE protein Vmw110 into cells prior to titration and by prior infection with ultraviolet light-inactivated herpes simplex virus. Mutant in1814 was essentially avirulent when injected into mice. The results demonstrate that transinduction of IE transcription by Vmw65 is important at low multiplicity of infection and in vivo but that at high multiplicity of infection the function is redundant.

Generation of mucosal mast cells is stimulated in vitro by factors derived from T cells of helminth-infected rats.

The connective tissue of rats, and several other species of mammals, contains two distinct types of mast cells that differ in morphology, histochemical staining properties and location1. One type, frequently called the normal connective tissue mast cell, can be obtained in nearly homogeneous preparation from a mixed cell population in the peritoneal cavity and forms the basis of our knowledge of mast cells. The other type is referred to as the mucosal mast cell because in normal rats it has been observed only in mucosal tissue. Infection with helminth parasites induces an exteNsive accumulation of mast cells and eosinophils in the tissues, and parasites of mucous surfaces, in particular, stimulate a rapid hyperplasia of mucosal mast cells. However, the origin of mucosal mast cells, and their relationship to the connective tissue mast cells is uncertain. We now slow that lymphocytes of helminth-infected rats, on in vitro stimulation with specific antigen, release factors causing pronounced mucosal mastocytosis in normal rat bone marrow cultures.