Outcomes of endoscopic ultrasound-guided pancreatic FNAC diagnosis for solid and cystic lesions at Manchester Royal Infirmary based upon the Papanicolaou Society of Cytopathology pancreaticobiliary terminology classification scheme.

OBJECTIVE: To compare endoscopic ultrasound (EUS)-FNAC diagnosis of pancreatic lesions with patient outcome based upon the Papanicolaou Society of Cytopathology pancreaticobiliary terminology classification scheme diagnostic categories: Panc 1 (non-diagnostic); Panc 2 (negative for malignancy/neoplasia); Panc 3 (atypical); Panc 4B (neoplastic, benign); Panc 4O (neoplastic, other); Panc 5 (suspicious of malignancy); and Panc 6 (positive/malignant). METHODS: All EUS-FNA pancreas specimens taken at Manchester Royal Infirmary in 2015 were prospectively classified according to the above scheme at the time of cytology reporting and data recorded prospectively. Subsequently, outcomes based on clinical follow-up or histopathology diagnosis were compared with the cytology diagnosis. RESULTS: 120 EUS-FNA pancreas specimens from 111 patients were received, of which 112 (93.3%) specimens had follow-up data. There were 79 and 41 EUS-FNA pancreas specimens from solid and cystic lesions, respectively. Based on the cytology diagnosis the specimens were classified as Panc 1 (7.5%), Panc 2 (33.3%), Panc 3 (2.5%), Panc 4B (2.5%), Panc 4O (15.0%), Panc 5 (3.3%) and Panc 6 (35.9%). The performance indicators for diagnosis of malignancy or neoplasia with malignant potential, included sensitivity (95.4%), specificity (100%), positive predictive value (100%), negative predictive value (92.3%), false positive rate (0%) and false negative rate (4.6%). CONCLUSIONS: The Papanicolaou Society of Cytopathology pancreaticobiliary terminology classification scheme is a logical system that can easily be introduced in a diagnostic cytopathology service. This classification scheme acts as an aid to diagnostic reporting, clear communication of significant results including risk of neoplasia/malignancy to clinicians, clinical audit and comparison of results with other centres.

Long-term follow-up of patients following negative colposcopy: a new gold standard and its implications for cervical screening.

From 1189 colposcopy referrals in 1997 at a single cervical screening centre, 88 women who had no biopsy taken at colposcopy (negative colposcopy) were identified. We followed up these women for a maximum of 4 years and calculated the positive predictive value (PPV) of a single smear before and after follow-up. Using slide review we attempted to correlate the grade of smear leading to colposcopy referral with final outcome. Our results showed that long-term follow-up alters the PPV of cervical cytology. Analysis showed a strong correlation between the review grade of the referring smear and the final outcome after follow-up. From these results we suggest an evidence-based protocol for cervical screening follow-up after negative colposcopy.

An alternate model of thyroid cytology: practical not perfect.

A sample of 384 thyroid cytology specimens prepared by cytospin over a 2.5-year period was classified by original report into inadequate, non-neoplastic and suspicious of neoplasia or worse. This was then compared with subsequent histology. The resulting data showed an inadequacy rate of 33%, a sensitivity of 55%, a specificity of 59%, a positive predictive value of 64% and a negative predictive value of 93%. On review of the cytology, in knowledge of the subsequent histology, the maximum achievable results were determined to have a positive predictive value of 79% and a negative predictive value of 97%. No clinically significant adverse event was detected.

Nucleotide sequencing and serological evidence that the recently recognized deer tick virus is a genotype of Powassan virus.

Deer tick virus (DTV) is a recently recognized North American virus isolated from Ixodes dammini ticks. Nucleotide sequencing of fragments of structural and non-structural protein genes suggested that this virus was most closely related to the tick-borne flavivirus Powassan (POW), which causes potentially fatal encephalitis in humans. To determine whether DTV represents a new and distinct member of the Flavivirus genus of the family Flaviviridae, we sequenced the structural protein genes and 5' and 3' non-coding regions of this virus. In addition, we compared the reactivity of DTV and POW in hemagglutination inhibition tests with a panel of polyclonal and monoclonal antisera, and performed cross-neutralization experiments using anti-DTV antisera. Nucleotide sequencing revealed a high degree of homology between DTV and POW at both nucleotide (>80% homology) and amino acid (>90% homology) levels, and the two viruses were indistinguishable in serological assays and mouse neuroinvasiveness. On the basis of these results, we suggest that DTV should be classified as a genotype of POW virus.

Amino acid substitution(s) in the stem-anchor region of langat virus envelope protein attenuates mouse neurovirulence.

The identification of variants that are unable to bind membrane receptor preparations (MRPs) has previously been shown to select attenuated yellow fever and Japanese encephalitis viruses. In this study, this methodology has been extended to the tick-borne serocomplex of flaviviruses. Langat (LGT) virus strain TP21 was bound to mouse or human brain MRPs and viruses that escaped binding were isolated and characterized. In addition, variant viruses escaping neutralization by the monoclonal antibody (MAb) 9F9 were also isolated. All of the variant viruses were attenuated for mouse neurovirulence (> or =13-fold). Sequence analysis of the prM/E region of the variant viruses identified mutations within the stem-anchor region of the E protein in variants isolated following incubation with mouse or human brain MRPs at a pH > or = 7.0. The MAb 9F9 variants and MRP variants isolated at pH 5.0, which should induce a conformational shift in the viral E protein, had nearly identical mutations in the prM/M protein immediately N-terminal to the prM/E cleavage site. MAb 9F9 neutralized none of the variant viruses and hemagglutination inhibition assays suggest that the variant virus surface proteins have slightly different conformations compared to the parental virus. These data support previous work indicating that the stem-anchor region of the E protein is important to the surface architecture of the tick-borne flaviviruses. In addition, this study demonstrates that the M protein is at least partially solvent accessible on the virion surface and that the M protein plays a role in maintaining the conformation of the M/E surface complex.

Langat virus M protein is structurally homologous to prM.

Langat (LGT) virus M protein has been generated in a recombinant system. Antiserum raised against the LGT virus M protein neutralizes tick-borne encephalitis serocomplex flaviviruses but not mosquito-borne flaviviruses, indicating that the M protein is exposed on the surface of virions. The antiserum recognizes intracellular LGT virus prM/M and binds to prM and M in Western blots of whole-cell lysates and purified virus, respectively. These data suggest that the prM and M proteins are structurally similar under native conditions and support the hypothesis that the "pr" portion of prM facilitates proper folding of the M protein for expression on the virion surface.

Nucleotide sequence and deduced amino acid sequence of the medium RNA segment of Oropouche, a Simbu serogroup virus: comparison with the middle RNA of Bunyamwera and California serogroup viruses.

The Bunyavirus genus of the family Bunyaviridae contains 18 serogroups. To date nucleotide sequence data has been obtained for three serogroups, Bunyamwera, California and Simbu, based on analysis of the small (S) RNA segment. In comparison, there is only nucleotide sequence data for the large and medium (M) RNA segments for members of the Bunyamwera and California serogroups. In this paper we report the nucleotide sequence of the M RNA of Oropouche (ORO) virus, a member of the Simbu serogroup. The M RNA was 4396 nucleotides in length with G1, G2 and NSm proteins similar in size to those reported for members of the Bunyamwera and California serogroups. However, there was limited nucleotide (50-52%) and amino acid (30-32%) homology between ORO virus M RNA and those of published members of the other two serogroups. The Bunyamwera and California serogroups are more closely related to each other than the Simbu serogroup virus Oropouche. These data were consistent with that previously reported for the S RNA (Saeed et al., 2000. J. Gen. Virol. 81, 743-748). It has been noted previously that three of four potential N-linked glycosylation sites of the Bunayamwera and California serogroups are conserved in G1 and G2 proteins. In contrast, ORO virus was found to have only three potential N-linked glycosylation sites of which only one, in G1, was conserved with members of the other two serogroups. Comparison of M RNA sequences of different strains of ORO virus revealed genetic variation consistent with that reported previously for the S RNA.

Thermodynamic mixing of molecular states of the epidermal growth factor receptor modulates macroscopic ligand binding affinity.

The epidermal growth factor receptor (EGFr), when expressed on the cell surface, has long been known to display two distinct affinities for epidermal growth factor (EGF) binding. In addition, the treatment of cells expressing the EGFr with phorbol esters has been shown to cause a loss of the high-affinity binding capacity of the receptor. In the present study, point mutations that alter acidic or phosphorylation sites have been made in an intracellular domain near Tyr-992 (residues 988-992) of the EGFr. Equilibrium (125)I-EGF binding studies demonstrate that the conversion of Tyr-992 into glutamate induces a 4-fold decrease in the EGFr apparent low-affinity dissociation constant, whereas the mutation of two acidic residues, Asp-988 and Glu-991, or the conversion of Tyr-992 into phenylalanine does not alter EGFr affinity. Phorbol ester treatment of EGFr-expressing Chinese hamster ovary cells results in a loss of high-affinity binding and an increase in the apparent low-affinity dissociation constant of the receptor, similar to the effect of a truncation mutant in which the C-terminal 190 residues are deleted. These results are examined in the context of a new model for regulation of the affinity of the EGFr for EGF in which a cytosolic particle stabilizes the high-affinity conformation of the EGFr and a rapid equilibrium exists between EGFr high-affinity and low-affinity conformations. This model demonstrates that the macroscopic affinities of the EGFr can differ from the affinities of individual EGFr molecules and provides a theoretical framework whereby the measured affinities of the EGFr are modulated by intracellular interactions.

What levels of agreement can be expected between histopathologists assigning cases to discrete nominal categories? A study of the diagnosis of hyperplastic and adenomatous colorectal polyps.

AIMS: To assess the levels of agreement between histopathologists for a two-class nominal categorization process--the discrimination between hyperplastic and adenomatous colorectal polyps. METHODS: Fifty hyperplastic and 50 adenomatous polyps received consecutively in the laboratory were categorized by nine histopathologists, and the level of agreement between all observers and the original diagnosis was assessed using kappa statistics. RESULTS: For the eight observers with 11 months or more experience in histopathology, there was a high level of agreement with kappa statistics ranging from 0.84 to 0.98. This process was performed rapidly with an average of 13 to 22 seconds spent on each case. One observer with only 6-weeks' experience of histopathology had a lower overall level of agreement with kappa statistics ranging from 0.46 to 0.54, but the performance on the later cases was much higher. CONCLUSIONS: The level of agreement in the distinction between hyperplastic and adenomatous colorectal polyps is high among histopathologists with at least moderate amounts of experience in histopathology. The one virtually naïve observer showed a marked learning response during the study without feedback on case outcome. This suggests that histopathologists are very reliable in assigning cases to distinct nominal categories and that learning of these processes occurs early in a histopathologist's career.

The French neurotropic vaccine strain of yellow fever virus accumulates mutations slowly during passage in cell culture.

This study of the yellow fever French neurotropic vaccine strain from the Institut Pasteur (FNV-IP) demonstrates that this viral genome is not as stable as that of the 17D-204 vaccine virus. FNV-IP was plaque-purified three times and then passaged eight times in Vero cells. Viral populations from the second and eighth passage post purification were sequenced and compared to the published sequences of FNV-IP. The passage-2 viral population had 31 nucleotide and nine amino acid changes compared to the parental virus while the passage-8 virus had six additional nucleotide changes encoding a single amino acid substitution. The plaque-purified virus also had two sequence deletions in the 3'-noncoding region. The plaque purification resulted in selection of a passage-2 virus that had a mouse LD(50) of 20 pfu/ml, 67-fold greater than parental FNV-IP which had an LD(50) of 0.3 pfu/ml. Subsequent passage in Vero cells resulted in a passage-8 virus which had increased neurovirulence with an LD(50) of 3.2 pfu/ml. The only amino acid difference between the passage-2 and passage-8 viruses was at amino acid 638 of NS5 which lies within domain V of the RNA-dependent-RNA polymerase. Overall, these data indicate that FNV-IP virus has an inherently less stable genome than 17D vaccine virus and a variable viral population.

Epidermal growth factor receptor internalization rate is regulated by negative charges near the SH2 binding site Tyr992.

This study examines the effects of mutations at and in the vicinity of tyrosine 992 of the epidermal growth factor receptor (EGFr) on epidermal growth factor- (EGF-) stimulated internalization of the receptor. Two regions of the EGFr adjacent to this domain have been defined previously as internalization domains. The present work shows that the mutation of negatively charged amino acid residues near Tyr992 to their uncharged analogues increases the rate of EGF receptor internalization. In addition, the conversion of Tyr992, which is an EGFr ligand-induced autophosphorylation site, to phenylalanine also increases the rate of receptor internalization. However, the mutation of Tyr992 to a glutamate residue does not alter the receptor internalization rate. In addition, the truncation of the EGFr at glutamate 996 reduces the internalization rate by half. This result confirms previous reports that residues immediately C-terminal to Glu996 are necessary to allow the normal rate of ligand-induced receptor endocytosis. The data suggest that negative charge in the vicinity of Tyr992, and potentially the phosphorylation of the EGFr at Tyr992, reduces the rate of ligand-induced receptor endocytosis. This reduction in internalization rate increases the lifetime of the activated EGFr in the plasma membrane by about 70%, thus suggesting that phosphorylation of Tyr992 acts to increase the signaling capacity of the EGF receptor even as it directly acts as an SH2 binding site.

Restrictions of T cell receptor beta chain repertoire in the peripheral blood of patients with systemic lupus erythematosus.

OBJECTIVE: To investigate whether oligoclonal T cell populations occur in peripheral blood lymphocytes from patients with systemic lupus erythematosus (SLE). METHODS: RNA was extracted from the lymphocytes isolated from whole peripheral blood of five female patients fulfilling ARA criteria for SLE and two healthy female controls, and synthesised into cDNA. CDR3 length spectratyping was performed using a polymerase chain reaction (PCR) run to saturation followed by a primer extension with a radioactively labelled primer. The resulting samples, one for each of the 23 V beta families, were then run on a polyacrylamide sequencing gel to examine the T cell receptor beta chain repertoire at the level of VDJ length heterogeneity. RESULTS: The two healthy female controls showed faint oligoclonal bands in only two and three V beta families respectively. Three of the patients showed a similar degree of oligoclonality to the controls, while the other two, who had active disease as shown by SLAM scores of 17 and 19 and in one case low C4 and raised C3dg levels, showed marked oligoclonality of their T cell beta chain repertoire affecting more than 17 of the 23 V beta families analysed. CONCLUSIONS: Using the technique of CDR3 length spectratyping, restriction of T cell receptor beta chain usage by peripheral blood T cells in patients with SLE has been demonstrated for the first time.

Stimulation of rat spleen cells by staphylococcal enterotoxins.

There is much interest in staphylococcal enterotoxins as T cell mitogens in humans, mice and rabbits. Rat spleen cells were shown to proliferate in response to staphylococcal enterotoxins A and B and toxic shock syndrome toxin-1 at concentrations (5 to 500 ng ml-1) which also stimulate mouse spleen cells. The proliferative response to all these enterotoxins was inhibited by cyclosporin A, indicating the response to be predominantly that of T cells. These results indicate that the rat provides another convenient model for the analysis of T cell responses to enterotoxins.