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To describe a case of papillary carcinoma of the thyroid with fibromatosislike stroma, emphasize the need for a diligent search for papillary thyroid cancer in the presence of a fibroproliferative lesion, highlight the peculiar hormonal response of the stromal component, and review the pertinent literature.We present the clinical, laboratory, radiologic, and pathologic findings in a patient with papillary carcinoma of the thyroid with fibromatosislike stroma and review the related published material.A 29-year-old woman presented to our surgical department because of a large mediastinal mass. She underwent surgical removal of the mass by means of a median sternotomy and neck extension. Pathology examination revealed macroscopically tan scarlike tissue, which by histologic study consisted of a dominant fibroproliferative lesion overshadowing a minor component of papillary carcinoma of the thyroid. Further neck exploration with total thyroidectomy revealed multifocal papillary carcinoma of the thyroid. Postoperatively, the patient received radioiodine treatment. A local and aggressive recurrent tumor was observed during a subsequent pregnancy; the lesion was not amenable to complete resection but fascinatingly responded to antiestrogen therapy (orally administered tamoxifen).The presence of a fibroproliferative lesion could be misleading. A diligent search should be made for a papillary thyroid carcinoma component within fibromatosislike stroma. The mode of manifestation of the tumor and its response to hormonal manipulation are distinctive features of this case.

Cardiac inflammatory myofibroblastic tumor (IMT) is a rare entity and is associated with distinct clinical, pathological and molecular features. The clinical behavior, natural history, biological potential, management and prognosis of such tumors are unclear. We present herewith an adolescent girl who presented with similar entity involving the junction of the right atrium and the inferior vena cava (IVC) in association with thrombocytosis and IVC thrombosis leading to obstruction of blood flow. Diagnostic tools included imaging and immuno-histopathology studies. Surgical management included resection of the tumor and thrombo-embolectomy of the IVC under cardiopulmonary bypass. This case is unique due to association of complete obstruction of IVC caused by the strategic location of the tumor, thrombosis of vena cava and association of thrombocytosis. These features have not been reported yet in relation to the cardiac IMT. This report will help in better understanding and management of similar cases in terms of planning cannulation of femoral veins or application of total hypothermic circulatory arrest during cardiopulmonary bypass and prompt us to look for recurrence or metastasis during follow up using echocardiography and laboratory investigations. The possibility of IMT should be kept in the differential diagnosis of cardiac tumors especially in children and adolescents.

Previous studies have shown that BRCA1-related breast cancers are often high-grade tumors that do not express estrogen receptors, HER2, p27Kip1, or cyclin D1, but do express p53 and cyclin E. In addition, the expression of cytokeratin 5/6 (CK5/6), indicating a basal epithelial phenotype, is frequent in BRCA1-related breast cancer. Here, in a series of 247 breast cancers, we demonstrate that CK5/6 expression was associated with nearly all of the features of BRCA1-related breast cancer and was also associated with a poor prognosis. In a parsimonious multivariable proportional hazards model, protein levels of cyclin E, p27Kip1, p53, and the presence of glomeruloid microvascular proliferation all independently predicted outcome after breast cancer. In this model, only cyclin E and p27Kip1 levels were independent predictors in lymph node-negative cancers, whereas glomeruloid microvascular proliferation and tumor size independently predicted outcome in node-positive disease. The molecular determinants of the basal epithelial phenotype encapsulate many of the key features of breast cancers occurring in germ-line BRCA1 mutation carriers and have independent prognostic value. Basal breast cancer deserves recognition as an important subtype of breast cancer.

Purified rubella virus treated with Triton X-100 was subjected to centrifugation in a sucrose density gradient containing nonionic detergent beta-D-octylglucoside. The result of this treatment was the formation of hemagglutinating rosettes containing viral glycoproteins VP2 (50,000 mol. wt.) and VP3 (63,000 mol. wt.). The rosettes have a 26 S sedimentation coefficient and a density of 1.2 g/cm3 in sucrose. Electron microscopy revealed 15 nm rosettes with a hollow center. The molecular weight of the rosettes was extrapolated at 850,000.

Background: The t(11;14)(q13;q32) translocation present in the majority of mantle cell lymphomas (MCLs) places the cyclin D1 gene under the control of immunoglobulin transcriptional regulatory elements, causing overexpression of cyclin D1. Quantification of cyclin D1 expression can distinguish MCL from other lymphomas. Methods: A quantitative real-time reverse transcription (RT)-PCR assay was developed for cyclin D1 mRNA suitable for use with RNA extracted from fresh and formalin-fixed, paraffin-embedded tissues. Specimens were amplified in an Applied Biosystems Model 7700 Sequence Detection System in reactions containing primers and probes for cyclin D1 and a control gene, β2-microglobulin. Relative expression of the two genes was standardized against a control MCL cell line, [M02058.][1] Results: The range of cyclin D1 expression among 20 MCLs was substantially higher than that in other lymphomas and reactive lymph nodes. By choosing an optimal cutoff point for assessing overexpression, the sensitivity and specificity of the assay for the diagnosis of MCL in lymph node specimens both approached 100%: Overexpression was detected in 20 of 20 MCLs, but in none of 21 non-mantle-cell lymphomas or 10 reactive lymph nodes. Conclusions: Quantitative real-time RT-PCR for cyclin D1 overexpression provides a rapid diagnostic test with clinical utility in the diagnosis of MCL. [1]: /lookup/external-ref?link_type=GEN&access_num=M02058.&atom=%2Fclinchem%2F47%2F2%2F195.atom

We have previously shown that intraperitoneal immunization of BALB/c mice with the 14 amino-acid long synthetic peptides G/174-187 and BG/174-187, representing the region 174-187 of the G-glycoprotein from human (H) and bovine (B) respiratory syncytial virus (RSV), respectively, completely protects animals from infection with the corresponding virus. A current goal in vaccine development being the delivery of noninvasive protective antigens via mucosal surfaces, we have evaluated the immunogenicity and protective efficacy of the two peptides when administered to mice by the intranasal (i.n.) route in the presence or absence of the cholera toxin (CT) as a mucosal adjuvant. The two peptides given alone induced the production of RSV-specific circulating IgG, as revealed by ELISA titers of immune sera. When the peptides were administered intranasally with CT, the higher IgG antibody titer which was induced was within the same order of magnitude as that obtained following i.n. immunization with live RSV or intraperitoneal injection with the peptides, thus demonstrating the stimulatory effect of the CT adjuvant. Moreover, although the peptides fail to induce a detectable level of secretory IgA, all animals immunized i.n. with peptide BG/174-187 (plus or minus CT) and all those immunized with peptide G/174-187 mixed with CT were completely resistant to infection by the corresponding virus. To our knowledge, this is the first study reporting that complete protection against a natural pathogen can be elicited by mucosally delivered synthetic peptides. This supports the usefulness of synthetic peptides in prophylactic vaccination.

N,N-((Butyloxy)propyl)amino diacetic acid is an amphoteric surfactant of the glycinate family. The synthesis of this glycinate was achieved via the ester route to obtain a pure compound. The infrared (IR) spectra of the aqueous solutions of this compound were obtained in the pH range 0.2−14 in order to determine the ionic distribution of the molecule as a function of pH. After subtracting the water bands in the IR spectra, factor analysis (FA) was used to separate the spectra of the ionic species and determine their abundance. The pKa's were retrieved from the volumetric titration as a function of pH. The values were used to calculate the volumetric abundance of each ionic species as a function of pH. The distribution curves thus obtained were compared with the normalized distribution curves obtained from the IR data. There is a good agreement between the two curves. The following species were obtained for the glycinate in water: the cation (pH 0−3.4), the zwitterion (pH 0−5.4), the monocarboxylate (pH 1...

The infrared (IR) spectrum of a sample in a saline solution cannot be retrieved adequately when the spectrum of pure water is subtracted. Some water bands remain in the spectrum. The retrieved spectrum is good only when the spectrum of NaCl in water at the right concentration is subtracted. However, when the concentration of the salt is unknown, it is still possible to obtain a good spectrum of the sample free of water. This is done by subtracting from the original spectrum a fraction of two eigenspectra of water: one for pure water and one for NaCl solvated water. The fraction of the eigenspectra is determined with the 2100 and 3300 cm−1 bands. The effectiveness of the method is illustrated with a solution of sodium monochloroacetate in a saline solution.

Previous reports demonstrate that synthetic peptides corresponding to the amino acid region 174-187 of G glycoprotein from subgroups A and B human respiratory syncytial virus (HRSV), containing a Cys-->Ser substitution at position 186, confer complete resistance to immunized BALB/c mice against infection with the respective virus. In this report, we show that a Cys186-->Ser substituted peptide (BG/174-187) representing the corresponding region of the bovine (B) RSV G glycoprotein conferred complete protection of mice against BRSV challenge, suggesting that the 174-187 region of RSV G glycoproteins constitutes a dominant protective epitope which has been maintained throughout evolution. Furthermore, immunization of calves with peptide BG/174-187 efficiently induced the production of antibodies capable of recognizing both the parental G glycoprotein and peptide BG/174-187. Following challenge with live BRSV, although none of the animals were protected from upper respiratory tract disease, there were little or no gross pneumonic lesions in the four peptide-immunized calves. In contrast, moderate to extensive pneumonic lesions were observed in 2 out of 3 calves in the control group. Our results thus suggest that peptide BG/174-187 efficiently prevented BRSV-associated pneumonia in the natural host. The use of this system as a model is quite promising with regard to the development of a human synthetic vaccine.

A 37-year-old female presented with Proteus syndrome and was found to have an asymptomatic enlarged spleen. Pathology confirmed splenic lymphangiomatosis. We describe an association of these 2 disorders in the Middle Eastern population. Diagnosis and pathogenesis are discussed in this case report.

The expression kinetics of an essential trans -regulatory protein, ICP27, from herpes simplex virus type 1 correspond to that of an immediate-early gene whose expression increases rapidly upon infection and then decreases as of 7 hr postinfection. In contrast, here we report that the bovine herpesvirus type 1 (BHV-1) homolog BICP27, a 50-kDa protein, is expressed as an early gene. Both the transcript and protein accumulated gradually reaching peak levels at approximately 12 hr postinfection, after which point steady state levels were maintained up to 24 hr. Thus the expression profiles of ICP27 and BICP27 are significantly different, suggesting that they may possess different functions.

Passive immunization of mice with 131 micrograms of the non-neutralizing monoclonal antibody (mAb) 18A2B2, directed against the A subgroup epitope of the G glycoprotein of respiratory syncytial virus Long strain (RSV), confers protection against viral i.n. challenge. The role of the Fc fragment of this antibody as well as the involvement of antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis towards protection was evaluated in vivo. Passive immunization with the Fab fragment alone (618-907 micrograms mouse-1) was unable to confer protection in mice. Furthermore, we passively immunized with the mAb 18A2B2 SCID beige mice, which are deficient in natural killer (NK) cell activity, to ascertain the role of NK cells in the protective mechanism. These mice were free of virus 5 days following viral challenge, indicating that NK cells do not contribute significantly towards the protective action of this antibody. Moreover, passively immunized BALB/c mice decomplemented with 8-10 U of cobra venom factor (CoVF) and DBA/2J mice (C5 deficient) were only partially protected. These findings suggest that in mice the alternative and classical pathways of the complement system are involved in the passive protection mechanism conferred by the non-neutralizing mAb 18A2B2. To our knowledge, it is the first description of a protective mechanism in mice that involves a non-neutralizing antibody and the complement system.

We previously reported the characterization of the gene encoding the bovine herpesvirus type 1 (BHV-1) major tegument protein VP8. With the aim of defining the immunological properties of this protein, we constructed a recombinant vaccinia virus (VV-VP8) in which expression of the VP8 gene was regulated by the P7.5 early/late promoter. Since the sequence of the VP8 gene contained a TTTTTNT motif known to serve as a transcription termination signal of vaccinia virus genes of the early class, a second recombinant (VV-VP8-Mut) in which this signal was modified by site-directed mutagenesis was created. Characterization of the recombinant viruses revealed that truncated VP8 mRNA and protein (69 kDa) were synthesized in VV-VP8 infected cells, whereas cells infected with VV-VP8-Mut produced a protein which was undistinguishable from that of the BHV-1 encoded protein (92–94 kDa). Immunization of BALB/c mice (H-2 d ) with VV-VP8-Mut induced a low VP8-specific antibody response whereas no specific response was induced in VV-VP8 inoculated mice. The low humoral response elicited was similar in C57BL/6 (H-2 b ) and C3H (H-2 k ) mice. Furthermore, immunization of mice with VV-VP8-Mut did not induce a BHV-1-specific lymphoproliferation in the three mice strains examined. Our results contrast with a recent study showing that immunization of calves with purified VP8 stimulated both T cell proliferation and antibody production.

Fluorescence spectra of hematoporphyrin IX (Hp) in water and in aqueous SDS solutions are obtained in the pH range 0.1 to 13 to determine the ionisation state of the molecule as a function of pH. In water, the spectra are complicated by aggregation which is quite severe near pH 4. In aqueous SDS, the aggregation is much less violent. Factor analysis (FA) is used to identify five species in the fluorescence spectra in each series of solutions. The distribution curve of these species as a function of pH is also obtained. By comparing the spectra and the distribution curve of Hp with those of HPPEEA, an ethanolamide derivative of Hp that does not contain the carboxylic groups (Part 3), the species are identified. For Hp in water we have obtained the following species: the dication in two allotropic forms in the pH range 0 to 5; the monocation (with the charge on an imino nitrogen) in the pH range 2 to 7; and the free base in the pH range 3.5 to 13. The monocation observed by the second derivative technique revealed three subspecies. For Hp in aqueous SDS we have obtained the following species: one dication in the pH range 0 to near 4; one monocation (with the charge on an imino nitrogen) in the pH range 0.5 to 9; three free bases (with no charge on the imino nitrogen) in the pH range 4 to 13. Of the latter, one species is the neutral molecule, another is a dianion (with the charges on the carboxylic side chains), and the third one appearing at pH higher than 10 is an allotropic form of the dianion.

Fluorescence spectroscopy of hematoporphyrin IX di-n-propylether diethanolamide (HPPEEA) in aqueous solutions, with and without SDS, was obtained in the pH range from 0.1 to 13. At pH greater than 3, HPPEEA in water solutions gives spectra complicated by aggregation whereas in aqueous SDS solutions, the aggregation is greatly reduced. Factor analysis is used to separate the spectra of the individual species from the experimental spectra. Five and four species are identified in pure water and in aqueous SDS solutions, respectively. The predominant species are: two free bases at pH higher than 6; one monocation at pH near 4; and two or one dications at pH lower than 2.5. The intensity signatures are related to the ionic distribution and to the aggregation situation of HPPEEA at different pH.

Chapados, C., Trudel M. and Miletic, J., 1994. Enhancing infrared spectra by Fourier deconvolution using the Cauchy—Gauss product function. Chemometrics and Intelligent Laboratory Systems , 22: 209–219. The method of Fourier deconvolution using Cauchy—Gauss product functions is being developed that allows the enhancing of experimental infrared spectra more adequately than with Cauchy (or Lorentz) functions. The mathematical development to obtain the Fourier transform of the product function is given. Two methods can be used to obtain the inverse Fourier transform of line shape functions: an analytical and a numerical method. The numerical method gives some errors which limit the degree of enhancement while the analytical method overcomes these difficulties. The numerical method is the only way to obtain an inverse Fourier transform of an experimental spectrum, and some aliasing is introduced by the algorithm used. The comparison between analytical and numerical methods of enhancing a simulated IR band is done to determine the limit of the procedure. The IR carbonyl band of methyl acetate is used as an example to show the effectiveness of the procedure described.

A method based on factor analysis is developed that permits us to retrieve the real spectra and multiplication factors (or concentrations) of individual species in spectra of evolving complex systems with no a priori information. The application of constraints of non-negative intensities and non-negative concentrations to the matrices of abstract factors gives the unoptimized real spectra. Then searching for the most likelihood spectra by using the maximum entropy criterion gives the real spectra. Mixtures of experimental spectra are used to illustrate the method.

Using in vitro translation of hybrid-selected mRNA, we have previously shown that bovine herpesvirus type 1 HindIII fragment M encodes an abundant 94K polypeptide. Using immunoprecipitation and sequencing analyses, it has now been shown that the polypeptide is related to the major tegument protein VP8 and is homologous to the herpes simplex virus type 1 major tegument proteins VP13/14. The sequence of the VP8 gene (field isolate 34) is reported and compared to published data. Several differences between the sequences were detected, resulting particularly from base insertions/deletions generating three major frameshifts affecting an area of 87 amino acid residues of the encoded protein. In addition, sequence comparison revealed 29 single base alterations, excluding frameshift regions, producing 17 amino acid substitutions. Overall, 14.1% of the deduced amino acid sequences were divergent. We have also established that the last 152 nucleotides of the previously reported sequence correspond to the sequence of the minus not the sense strand. Finally, we report that the 4.4 kb transcript of the VP8 gene is initiated 39 nucleotides upstream from the translation start codon.

The complete nucleotide sequence of the gene encoding the small subunit of ribonucleotide reductase (RNR) from bovine herpesvirus type-1 (BHV-1) was determined. The genomic DNA fragment sequenced also represented regions corresponding to the carboxy termini of RNR large subunit and of a virion protein causing host shut-off. The small subunit polypeptide was constituted of 314 amino acid residues totalling 35.25 kDa. The major transcription initiation and termination sites of the small subunit mRNA were located 95 bases upstream and 88 nucleotides downstream from the coding region, respectively. These findings indicate that the mRNA was 1128 bases long which correlated well with the size of the polyadenylated transcript detected in Northern blot analysis (1.3 kb). Within the RNR large subunit coding region, a TATA box and two CAAT box motifs were found 26, 104, and 190 nucleotides, respectively, upstream from the transcription initiation site of the small subunit mRNA. In contrast to previous studies (Slabaugh et al., J. Virol. 1988, 62, 519-527; Boursnell et al., Virology 1991, 184, 411-416), our comparative analysis of five herpesviruses, one iridovirus, and one poxvirus small subunit protein sequences suggested that the seven viruses arose from a common lineage.

Respiratory syncytial virus is an important human pathogen causing serious lower respiratory tract infections of children and elderly people. Previous studies on the development of experimental subunit vaccines either expressed by recombinant DNA technology or prepared from purified viral proteins absorbed on adjuvant (ISCOMs) have shown promise. The present work reports on the effectiveness of an experimental ISCOMs vaccine in initiating humoral and cell-mediated immune responses and in providing overall protection upon live virus challenge in Balb/c mice; results indicate that vaccination by the intramuscular route is more effective, even if vaccination by the intranasal route also significantly reduced virus shedding.

A synthetic peptide homologous to amino acids 174-187 of the G glycoprotein of the A2 strain of human respiratory syncytial (RS) virus (G/174-187) was shown to induce protection from live virus challenge of BALB/c mice after immunization with three doses of 50 micrograms of peptide coupled to keyhole limpet hemocyanin. Immunized mice showed high levels of circulating RS-specific antibodies as detected by ELISA assay; however, no neutralizing antibodies were found. Moreover, an important short-term cytotoxic T-cell response was observed with lymphocytes isolated from the lungs but not from the spleen of immunized mice. This response was lost 24 weeks after immunization; however, mice remained protected against challenge with live RS virus. In addition, a monoclonal antibody that specifically binds to peptide G/174-187 was found efficient in conferring passive protection from challenge: this data further supports our results on the importance of the 174-187 region in protection. Another peptide, spanning amino acids 144 to 159, was shown to induce neutralizing antibodies but did not confer protection.

We have previously located a major neutralization site of the fusion protein of respiratory syncytial virus (RSV) in the polypeptide region extending from amino acids Ile221 to Glu232. In this report, 8 peptides corresponding to the six major hydrophilic regions of the F1 subunit were selected to analyse their immunogenic and protective capacities as well as their ability to block the high neutralization activities of 4 monoclonal antibodies (MAbs). Only 5 of the 8 peptides tested induced specific antibodies while all induced an in vitro interleukin-2 response of splenocytes from immunized mice. Peptide 3 (Ile221-Phe237) was able to elicit neutralizing antibodies, confirming our previous hypothesis concerning the location of a neutralization site. However, immunization with the latter did not induce significant reduction of virus in lungs of BALB/c mice upon challenge, probably due to an inadequate level of circulating neutralizing antibodies. Interestingly, peptides 2 (Asn216-Glu232), 3 (Ile221-Phe237), and 5 (Ser275-Ile288) blocked in vitro neutralization by four different F1 specific MAbs. A hypothesis is proposed to explain these results.

A basal epithelial phenotype is found in not more than 15% of all invasive breast cancers. Microarray studies have shown that this phenotype is associated with breast cancers that express neither estrogen receptor (ER) nor erbB-2 (HER2/neu) (i.e., ER/erbB-2-negative tumors). The ER/erbB-2- negative phenotype is also found in breast cancers occurring in BRCA1 mutation carriers (i.e., BRCA1-related breast cancers). We tested the hypothesis that BRCA1-related breast cancers are more likely than non-BRCA1/ 2-related breast cancer to express a basal epithelial phenotype. Among 292 breast cancer specimens previously analyzed for ER, erbB-2, p53, and germline mutations in BRCA1 and BRCA2, we identified 76 that did not overexpress ER or erbB-2. Of the 72 specimens with sufficient material for testing, 40 expressed stratified epithelial cytokeratin 5 and/or 6 (5/6). In univariate analysis, the expression of cytokeratin 5/6 was statistically significantly associated with BRCA1-related breast cancers (odds ratio = 9.0, 95% confidence interval = 1.9 to 43; P =.002, two-sided Fisher's exact test). Thus, germline BRCA1 mutations appear to be associated with a distinctive breast cancer phenotype.

Background: Patients with leukemia often manifest cutaneous findings, which include nonspecific lesions and specific leukemic infiltrates termed leukemia cutis. Objective: A case of leukemia cutis involving distal finger pads is reported and literature describing hand involvement of specific leukemic infiltrates is reviewed. Methods and Results: An 80-year-old woman with a 10-year history of chronic lymphocytic leukemia developed painful symmetric tumors of her distal finger pads. Histopathological examination of the biopsy specimen revealed infiltration by neoplastic lymphocytes. Only a few cases of leukemia cutis involving the hands have been reported in the literature, none with this particular presentation. The clinical and histopathologic features of leukemia cutis are reviewed. Conclusion: This case emphasizes the importance of obtaining a biopsy specimen for histopathological examination of any suspicious skin lesion in a patient with leukemia.

Human respiratory syncytial virus (RSV) is the primary cause of respiratory infection in infants worldwide. Currently there is no available vaccine, although studies in animal models have demonstrated protective immunity induced by an epitope of the RSV G-protein representing amino acids 174-187. Two peptides containing amino acids 174-187 of the G-protein of the human RSV A2 strain (NF1-RSV/172-187 and NF2-RSV/170-191) were separately engineered as translational fusions with the alfalfa mosaic virus coat protein and individually expressed in Nicotiana tabacum cv. Samsun NN plants through virus infection. RSV G-protein peptides were expressed in infected plant tissues at significant levels within 2 wk of inoculation and purified as part of recombinant alfalfa mosaic virions. BALB/c mice immunized intraperitoneally with three doses of the purified recombinant viruses showed high levels of serum antibody specific for RSV G-protein and were protected against infection with RSV Long strain.

A subunit approach to the development of a respiratory syncytial virus (RSV) vaccine was investigated. It involved the production, inEscherichia coli,of an RSV (Long) G protein fragment (G2Na) as a C-terminal fusion partner to an albumin binding region (BB) of streptococcal protein G. G2Na incorporated amino acid residues 130–230 and was specifically recognized by murine anti-RSV-A polyclonal serum. In mice, intraperitoneal immunization with BBG2Na induced high anti-RSV-A serum ELISA titers and low to moderate neutralization activity. The immune response induced by BBG2Na demonstrated a potent protective efficacy against upper and lower respiratory tract RSV-A infection. The immunogenicity and protective efficacy of BBG2Na was maintained for at least 47 and 48 weeks, respectively, and was as potent and durable as live RSV-A administered in a similar fashion. Intramuscular immunization of cotton rats with BBG2Na protected lungs from both homologous and heterologous virus challenge. In contrast to mice, however, cotton rat nasal tracts were not protected after BBG2Na immunization. Consistent with antibody-mediated protection, virus was cleared within 24 hr from the lungs of BBG2Na-immunized mice. The anti-RSV-A antibodies induced in mice were exclusively of the IgG1 isotype and were detected in the serum, lungs, and nasal tracts. Passive transfer of these antibodies prevented acute, and eliminated chronic, RSV-A lung infection in normal and immunodeficient mice, respectively, confirming that such antibodies are important and sufficient for BBG2Na-induced pulmonary protection. Our results clearly demonstrate that BBG2Na contains an important immunogenic domain of the RSV G protein. The prokaryotic origin of this protein indicates that glycosylation of the RSV G protein is not necessary for protective efficacy. Thus, BBG2Na has potential as an RSV subunit vaccine.

We identified subgroup specific protective epitopes represented by the amino acid regions 174–187 and 171–187 of the G glycoproteins from respiratory syncytial virus (RSV), subgroups A and B. Mice immunized with coupled synthetic peptides corresponding to either the region 174–187 containing a Cys186 → Ser substitution or to the native region 171–187 were completely resistant to RSV infection but only to the respective virus. The protective activities of the peptides 174–187 were dependent on the Cys186 → Ser substitution. In addition, a recombinant protein representing the region 125–203 of the A subgroup G glycoprotein expressed in Escherichia coli was capable without further treatment to completely protect animals against RSV subgroup A infection. We show that the combinations of cysteinyl residues (positions 173, 176, 182, and 186) retained within either synthetic peptides or the recombinant protein G 125–203 greatly influenced their protective activities. This indicates that the region 171–187 is essential for the protection conferred by the G 125–203 protein. Furthermore, our results strongly suggest that the peptides' and recombinant protein's potencies are a function of a loop-like structure which is stabilized by intramolecular disulfide linkages between Cys176–Cys182 and Cys173–Cys186. This is further supported by the observation that chemical blocking of the sulfidryl groups in synthetic peptides completely eliminated their protective activity.

We examined 100 breast cancers for retinoblastoma (Rb) and p53 protein expression by immunohistochemistry using the PMG3.245 and PAb 1801 antibodies. We assessed percentages of reactive cells and their intensity, as well as staining patterns. The results were correlated with neu protein reactivity and a panel of variables, including age, tumor size and type, nuclear grade, estrogen receptor/progesterone receptor content, and lymph node status. Retinoblastoma protein negativity, either partial or complete, was noted in 47% of cases. Surprisingly, a relatively stronger Rb reaction was seen in some high nuclear grade tumors. p53 positivity was found in 23% of cases and was a significant predictor of Rb loss. p53 also was correlated with poorly differentiated (nuclear grade III) neoplasms and neu expression but not with negative ER status. Tissue distribution profiles for Rb-negative and p53-positive cells were variable in this series, with both uniform and heterogeneous patterns observed. This suggests that Rb and p53 alterations may represent early or late events in transformation. Our findings further implicate Rb and p53 derangements in mammary oncogenesis.

Two antigenic sites recognized by neutralizing monoclonal antibodies (MAbs) directed against the fusion (F) glycoprotein of human respiratory syncytial virus were mapped on the primary structure of the protein by (i) the identification of amino acid substitutions selected in antibody-escape mutants and (ii) the reactivity of synthetic peptides with MAbs. The first site contained several overlapping epitopes which were located within the trypsin-resistant amino-terminal third of the large F1 subunit. Only one of these epitopes was faithfully reproduced by a short synthetic peptide; the others might require specific local conformations to react with MAbs. The second antigenic site was located in a trypsin-sensitive domain of the F1 subunit towards the carboxy-terminal end of the cysteine-rich region. One of these epitopes was reproduced by synthetic peptides. In addition, mutagenized F protein with a substitution of serine for arginine at position 429 did not bind MAbs to the second site. These results are discussed in terms of F protein structure and the mechanisms of virus neutralization.

We have analyzed viral DNA patterns, obtained following digestion of six different infectious bovine rhinotracheitis isolates, using a panel of restriction enzymes. Several differences in profiles were observed, particularly for a Canadian viral strain which presumably contained a genome that was larger than the other five infectious bovine rhinotracheitis genomes. Also, an insertion/deletion of a HindIII restriction site was found in the DNA of a Danish strain; this additional HindIII site was localized precisely within the original HindIII E fragment of the infectious bovine rhinotracheitis virus. Interestingly, results could be used to distinguish each viral strain examined from the others by simple digestion with the appropriate enzyme.

A bovine herpesvirus I (BHV-I) HindIII genomic bank spanning 89% of the entire genome was constructed and individual fragments analyzed for their capacity to select specific mRNAs which were then expressed by in vitro translation assays. This procedure allowed the mapping of more than 20 viral polypeptides to discrete regions of the DNA genome. Some polypeptides map in neighboring HindIII fragments while most seem encoded in single fragments. In particular, the coding sequences for an abundant 94 kDa polypeptide, which is the potential unglycosylated precursor of gII glycoprotein, have been assigned to the small 3.6 kbp HindIII genomic fragment M. The localization of structural and non-structural gene-coding sequences will help to characterize viral polypeptides and eventually, a better understanding of BHV-I infection will be gained.

The efficiency of vaccination via the mucosal route of an experimental human respiratory syncytial (RS) virus ISCOMS subunit vaccine based on the fusion protein of RS was evaluated. The circulating neutralizing antibody response was monitored when vaccine was inoculated intranasally in comparison with a parenteral route (intramusculaly); 105 infectious units of RS Long strain virus were used as live virus control. Results indicate the feasibility of intranasal vaccination with the ISCOMS.

A basal epithelial phenotype is found in not more than 15% of all invasive breast cancers. Microarray studies have shown that this phenotype is associated with breast cancers that express neither estrogen receptor (ER) nor erbB-2 (HER2/neu) (i.e., ER/erbB-2-negative tumors). The ER/erbB-2- negative phenotype is also found in breast cancers occurring in BRCA1 mutation carriers (i.e., BRCA1-related breast cancers). We tested the hypothesis that BRCA1-related breast cancers are more likely than non-BRCA1/ 2-related breast cancer to express a basal epithelial phenotype. Among 292 breast cancer specimens previously analyzed for ER, erbB-2, p53, and germline mutations in BRCA1 and BRCA2, we identified 76 that did not overexpress ER or erbB-2. Of the 72 specimens with sufficient material for testing, 40 expressed stratified epithelial cytokeratin 5 and/or 6 (5/6). In univariate analysis, the expression of cytokeratin 5/6 was statistically significantly associated with BRCA1-related breast cancers (odds ratio = 9.0, 95% confidence interval = 1.9 to 43; P =.002, two-sided Fisher's exact test). Thus, germline BRCA1 mutations appear to be associated with a distinctive breast cancer phenotype.

Competition experiments and biological assays with a panel of 15 monoclonal antibodies confirmed the presence of at least four antigenic sites on the fusion protein of human respiratory syncytial virus, three of which were involved in virus neutralization. One antigenic site, recognized by two strongly neutralizing antibodies, was conserved after reduction and denaturation and shown by immunoblotting to be localized on the F1 fragment of the fusion protein. Cleavage of this protein with staphylococcal protease V8 or papain produced a series of smaller peptides from 11 to 7 kilodaltons that retained this important neutralization determinant. Compared with the other neutralization sites, the epitope defined by monoclonal antibody 7C2 thus appears as the major neutralization epitope. Our peptide mapping results support the hypothesis that this major epitope is composed of a continuous sequence on the viral genome.

Rubella virosomes were prepared from preformed liposomes and detergent solubilized viral hemagglutinin. The liposomes were made from lecithin/dicetyl phosphate (3.5: 1) films resuspended in NTE buffer and sonicated. Viral hemagglutinin was prepared from purified virus after solubilization with Triton X-100 and centrifugation through a sucrose gradient containing β- d -octylglucoside. Electron microscopy of the rate zonal purified virosomes, showed virus-like structures of 40–80 nm. The virosomes retained the biological activity of the hemagglutinin and had a buoyant density of 1.2 g/cm 3 .

Neutralizing monoclonal antibodies were produced against a reference vaccine or a reference wild strain of poliovirus type 1, 2, and 3. After 26 fusions, 55 monoclonal antibodies were obtained with serotype 1 as the immunizing antigen, 180 with serotype 2, and 115 with serotype 3. The neutralizing activity of these monoclonal antibodies was tested first with the two reference strains and then if reactive, against a panel of 10 well-characterized strains of each serotype, 5 vaccinelike (VL) and 5 non vaccinelike (NVL). All monoclonal antibodies were type specific without reactivity with any of the heterologous strains. There was a wide range of reactivity within the strains of each serotype. Several monoclonal antibodies to serotype 1 reacted with all type 1 strains, while several neutralized strongly all VL strains and weakly one or more of the NVL strains. Most of the 180 monoclonal antibodies to serotype 2 neutralized to various degrees all strains of this serotype and about half reacted very strongly with all homologous strains whether VL or NVL. None could differentiate all VL and NVL homologous strains. Of the 115 monoclonal antibodies to serotype 3, several monoclonal antibodies neutralize to various levels all homologous strains and some can differentiate VL and NVL strains.Key words: poliovirus, monoclonal antibody, neutralization, serodifferentiation.

A microassay, using tissue culture microplates for the titration of human respiratory syncytial virus by syncytium formation, is described. Virus titers obtained agreed well with those obtained in a larger assay system; the microassay, however, is more rapid and economical. Large numbers of virus samples are easily and rapidly processed as the assay necessitates an incubation period of only three days.

An enzyme-linked immunosorbent assay (ELISA) for the identification and typing of polioviruses is described. Polioviruses could be rapidly detected and typed from cell culture supernatants by a double antibody sandwich technique. The assay is valuable for the rapid screening of a large number of viral isolates from water samples submitted for virological analysis. Several hundred isolates per day can be typed and only the remainder have to be tested by the conventional serum neutralization test.

Summary The immunoelectron microscopy (IEM) technique has been used for the detection of a rabbit enteric coronavirus (RECV). Immune serum was prepared in guinea pigs; the viral antigen used for the immunization procedure was obtained from the caecum of a sick rabbit, concentrated by centrifugation and purified on Percoll gradient. In order to identify the viral particles used in the immunization procedure, the protein pattern of the particles was determined by electrophoresis and compared with the pattern of other known coronaviruses. Analysis of structural polypeptides of the purified viral particles revealed a pattern similar to that reported for other coronaviruses. These polypeptides cross reacted with two other coronavirus specific immune sera (IBV and TGE). IEM assay of fecal samples collected from healthy and sick rabbits showed the presence of immune aggregates in specimens from both sick and healthy rabbits. Those aggregates contained viral particules sharing morphological characteristics with other coronaviruses. Furthermore, IEM assay was shown to be more sensitive than a direct EM procedure to detect coronavirus particles in rabbit feces. This assay also allowed the detection of a larger number of chronic carriers.

Individual rubella virus structural polypeptides were electroeluted from SDS-polyacrylamide gels. The eluted polypeptides were used, without further purification, as antigens in ELISA assays for the detection of rubella-specific antibodies in patients' sera. This provided a more sensitive detection method than that involving classical serological assays such as HI or VN or that using immunoprecipitation. Antisera against individual viral polypeptides were raised in mice. No haemagglutination inhibition activity was observed in any of these sera and weak virus neutralizing activity was only detected with antiserum to the E 1 protein. Antisera to either the E 1 or E 2 (a,b) complex proteins cross-reacted with both the E 1 and E 2 (a,b) complex proteins.

An indirect immunoperoxidase micro-assay, using a continuous cell line derived from ovine kidney cells (OK) and a previously characterized monoclonal antibody (7C2), specific for an exposed and highly conserved epitope of the fusion protein of different strains of RS virus, was used advantageously to rapidly titrate bovine, caprine and human strains of RSV by either quantal (TCID50) or plaque forming assays. Virus titers, obtained in less than 36 h, were in agreement with those obtained by the conventional plaque assays which required an incubation period of 4 days or more. This assay is also applicable to micro-neutralization of fusion inhibition assays for testing serum or screening monoclonal antibodies.

The intracellular and structural polypeptides of the Los Angeles and Cooper 1 reference strains of bovine herpesvirus 1, together with 12 other Canadian field isolates, were analyzed by polyacrylamide gel electrophoresis. Although a few minor differences were noted among some isolates in regard to intracellular viral protein content, analysis of partly purified virus showed strikingly similar polypeptide profiles among 19 proteins with molecular masses of 14 to 145 kilodaltons (kDa). Moreover, a neutralizing monoclonal antibody produced against the Cooper 1 strain also neutralized all of the other 13 strains tested in this study and immunoprecipitated the major 90-kDa glycoprotein. A second monoclonal antibody with a high hemagglutination inhibition titer prevented hemagglutination of other strains tested and also reacted against the 90-kDa glycoprotein by immunoprecipitation, indicating that this glycoprotein is responsible for the hemagglutinating activity of the viral particle and carries an important neutralization epitope.

A collaborative virological survey of drinking water was initiated in three major Canadian urban areas, Montreal, Ottawa, and Toronto. In each selected area, three water purification plants were sampled monthly for up to 18 months. The total population served by all nine plants was about 1 500 000. Samples of raw (100 L) and treated (1000 L) water were examined by virus concentration procedures based on adsorption–elution. Sample concentrates were assayed for cytopathic viruses on BS-C-1 cells and the results were expressed as the most probable number of cytopathic units (MPNCU). Viruses were detected in 57% (0–15.35 MPNCU/L) of the raw water samples from Montreal, 37% (0–46.0 MPNCU/L) in Ottawa, and 33% (0–4.91 MPNCU/L) in Toronto. The majority of isolates were reoviruses, but picornaviruses were also found. All finished waters (177 samples) met bacteriological, turbidity, and residual chlorine standards and were free of detectable viruses.

Using C57BL/HPB mouse erythrocytes, hemagglutination has been observed with the Los Angeles and Colorado-1 strains of bovine herpesvirus type 1 and with 12 other Canadian field isolates as well. The specificity of the hemagglutination observed with the viral strains has been confirmed by a hemagglutination-inhibition assay.

Rubella virus was purified by chromatography on Sepharose 2B after concentration by ultrafiltration on hollow fibers and hydroextraction with PEG 20,000. Yields of 40% infectivity and 70% hemagglutinating activity were routinely obtained. Chromatographie purification was compared to ultracentrifugation in sucrose, metrizamide and sodium metrizoate. Yields were lower in sucrose and metrizamide, while sodium metrizoate reduced the infectivity of the virus below detectable levels. These results demonstrate the advantage of Sepharose 2B for the purification of infectious rubella virus.

Several computer enhancement techniques are compared for their ability to resolve overlapping infrared carbonyl bands of chlorophyll obtained in the solid state. These methods are: the band fit optimization technique, the second-derivative technique, the subtraction of a super-smooth spectrum technique, and the Fourier self-deconvolution technique. The advantages and disadvantages of each technique are compared. In order to extract the maximum information on the spectra, two techniques should be used. The best combination we found was to first use the Fourier self-deconvolution technique, which gives some information on the number and the position of the bands, and—with this information—to then use the band fit optimization technique, which gives the exact intensity, bandwidth, and band shape, as well as the integrated intensity of all the bands.

Trypsinized normal rat embryo fibroblasts and untrypsinized and trypsinized transformed rat fibroblasts have two orders of binding sites for bacterial glycolipid mR595. The high order sites fix 1--3 micrograms glycolipid mR595/10(5) cells and those of the low order fix about 6 microgram glycolipids mR595/10(6) cells. Ca++ is required for the low order glycolipid mR595 binding to be trypsinized but not to the untrypsinized transformed rat fibroblasts. The low order binding is temperature dependent with the transition temperature lying between 25 and 37 degrees C. Exogenously added ganglioside and glycoproteins contained in the fetal calf serum do not inhibit fixation of glycolipid mR595. Only beta-lipoprotein at high concentrations is slightly inhibitory. Glycolipid mR595 fixation to transformed fibroblast does not alter their morphology and appears to slightly improve cell attachment to substratum. Glycolipid mR595 fixation results in a lengthening of the S-phase of the cell cycle and a reduction in 2-deoxyglucose uptake. Uptake of inorganic phosphate is not affected. Inhibition of phospholipid synthesis is observed in mR595 fixed fibroblasts whereas synthesis of cell surface glycoproteins and the content of cellular gangliosides is not affected.