B3 Subtype Research Articles

Ultrasonographic prevalence and proposed morphologic classification of bilobed gallbladder in cats.

Morphologic anomalies of the feline gallbladder (GB) have been previously reported in the literature. These morphologic variants are frequently encountered on routine abdominal ultrasound examination. The aim of this study was to provide an ultrasonographic classification system of these variants and document the overall incidence in the feline population. A prospective, descriptive study was undertaken; cats that had an abdominal ultrasound examination that included at least one sagittal and transverse plane image of the GB were included. GB shape was evaluated and categorized based on a classification scheme of morphologic variants modified from the human literature. Septated (S), bilobed (B1, B2, B3), duplex (D) and complex (C) categories were described. Of 516 cats included in the study, 389 had normal GB morphology, while 127 had anomalous GB morphology. The overall incidence rate of anomalous GB morphology was 24.61%. When examined by morphologic type, the septated (S) morphology had an incidence of 9.69%. A bilobed (B) morphology was the most commonly observed classification; incidence was 14.35% within our population; incidence of B1, B2 and B3 subtypes were 2.91%, 6.98% and 4.46%, respectively. Duplex GBs only made up 0.39% of the total population. The incidence of complex (C) morphologies was 0.19%. The incidence of GB morphologic anomalies was higher in our population than previously reported. Identification of these anomalies on routine ultrasound evaluation is common; numerous different morphologies can be identified and a standardized classification scheme is proposed. Complete evaluation of morphology can be challenging, particularly with regard to cystic duct anatomy. Clinical significance is uncertain and future studies are warranted to determine the relationship between morphologic variants and hepatobiliary disease.

Micro-Droplet Platform for Exploring the Mechanism of Mixed Field Agglutination in B3 Subtype.

B3 is the most common subtype of blood group B in the Taiwanese population, and most of the B3 individuals in the Taiwanese population have the IVS3 + 5 G > A (rs55852701) gene variation. Additionally, a typical mixed field agglutination is observed when the B3 subtype is tested with anti-B antibody or anti-AB antibody. The molecular biology of the gene variation in the B3 subtype has been identified, however, the mechanism of the mixed field agglutination caused by the type B3 blood samples is still unclear. Therefore, the purpose of this study was to understand the reason for the mixed field agglutination caused by B3. A micro-droplet platform was used to observe the agglutination of type B and type B3 blood samples in different blood sample concentrations, antibody concentrations, and at reaction times. We found that the agglutination reaction in every droplet slowed down with an increase in the dilution ratio of blood sample and antibody, whether type B blood or type B3 blood was used. However, as the reaction time increased, the complete agglutination in the droplet was seen in type B blood, while the mixed field agglutination still occurred in B3 within 1 min. In addition, the degree of agglutination was similar in each droplet, which showed high reproducibility. As a result, we inferred that there are two types of cells in the B3 subtype that simultaneously create a mixed field agglutination, rather than each red blood cell carrying a small amount of antigen, resulting in less agglutination.

Reliability of the Penny and Beit CURE Radiologic Classifications of Pediatric Patients with Chronic Hematogenous Osteomyelitis in the Philippine General Hospital

Objective. This study aimed to evaluate the inter- and intraobserver reliability of the Penny and Beit CURE radiologic classifications of pediatric patients with Chronic Hematogenous Osteomyelitis (CHOM) in the Philippine General Hospital (PGH).
 Methods. Thirty-four pre-operative radiographs of PGH pediatric patients with CHOM were classified by seven orthopedic surgeons using both Penny and Beit CURE Classification systems. Two sets of radiographs were sent to the surgeons twice, four weeks apart, to classify. The Fleiss and Cohen κ statistics were used to determine inter- and intraobserver reliabilities, respectively.
 Results. The Penny Classification had a slight to fair interobserver reliability (Fleiss κ = 0.17 and 0.24) and a fair intraobserver reliability (Cohen κ =0.35) with a 49.58% average intraobserver agreement. The interobserver reliability when including all Beit CURE classification subtypes was fair (κ = 0.28 and 0.31). This improved to moderate (κ = 0.41 and 0.54) when using only the four main types of the Beit CURE classification with a 77.31% intraobserver agreement.
 Conclusion. The Beit CURE classification for pediatric CHOM had higher inter- and intraobserver agreement rates than the Penny classification. Further improvement in reliability can be made by combining B2 and B3 subtypes under the Beit CURE classification.

Is the AO spine thoracolumbar injury classification system reliable and practical? a systematic review

Controversy surrounding the classification of thoracolumbar injuries has given rise to various classification systems over the years, including the most recent AOSpine Thoracolumbar Injury Classification System (ATLICS). This systematic review aims to provide an up-to-date evaluation of the literature, including assessment of a further three studies not analysed in previous reviews. In doing so, this is the first systematic review to include the reliability among non-spine subspecialty professionals and to document the wide variety between reliability across studies, particularly with regard to sub-type classification. Relevant studies were found via a systematic search of PubMed, EBESCO, Cochrane and Web of Science. Data extraction and quality assessment were conducted in line with Cochrane Collaboration guidelines. Twelve articles assessing the reliability of ATLICS were included in this review. The overall inter-observer reliability varied from fair to substantial, but the three additional studies in this review, compared to previous reviews, presented on average only fair reliability. The greatest variation of results was seen in A1 and B3 subtypes. Least reliably classified on average was A4 subtype. This systematic review concludes that ATLICS is reliable for the majority of injuries, but the variability within subtypes suggests the need for further research in assessing the needs of users in order to increase familiarity with ATLICS or perhaps the necessity to include more subtype-specific criteria into the system. Further research is also recommended on the reliability of modifiers, neurological classification and the application of ATLICS in a paediatric context.

Abstract PS7-52: The upgrade risk to (pre-)invasive breast cancer for B3 lesions diagnosed on core needle or vacuum assisted biopsy. A Belgian retrospective study

Abstract Introduction: Flat epithelial atypia (FEA), classical lobular neoplasia (LN), papillary lesions (PL), radial scars (RS) and atypical ductal hyperplasia (ADH) are lesions of uncertain malignant potential in the breast, which are described as B3 lesions in the B classification system of the European Working Group for Breast Cancer Screening Pathology. Current standard for management of B3 lesions on core needle biopsy (CNB) or vacuum assisted biopsy (VAB), is wide local excision (WE). However, recent consensus-based guidelines no longer recommend WE for all such lesions, but propose surveillance following CNB or VAB to be sufficient in some cases. In the absence of a Belgian guideline on the treatment of B3 lesions, this study was conducted to identify which B3 lesions have the lowest likelihood for breast malignancy and could therefore be spared from WE. Methods: Using data from the Belgian Cancer Registry (BCR), all patients with a new diagnosis of a B3 lesion on CNB or VAB between 2013-2016 and who had a histological follow up with VAB or WE after CNB or WE after VAB were included. Histological follow-up was retrieved from BCR and limited to 12 months following diagnosis. Histology was compared between the first- and follow-up investigation to determine the upgrade risk to ductal carcinoma in situ (DCIS) or invasive breast cancer (IC) according to the type of B3 lesion. Patients with synchronous (pre-) invasive lesions were excluded. Results: Between 2013-2016 there were 812 B3 lesions available for upgrade analysis after initial diagnosis. After CNB 551 lesions had WE or VAB as follow up and after VAB 261 lesions had WE. After primary diagnosis on CNB, the total upgrade risk was 19,0%. There was histological agreement in 57,9% and no B3 lesion or upgrade was reported in 21,8%. Per B3 lesion subtype the upgrade risk to DCIS - IC after diagnosis on CNB was: ADH 17,1%-12,4%, FEA 21,1% - 18,4%, LN 18,9% -21,6%, RS 14,3% - 11,4%, and PL 7,2% - 3,2%. After initial diagnosis on VAB the total upgrade risk was 14,9%. There was histological agreement in 52,9% and no B3 lesion or upgrade was found in 31,4%. Per B3 subtype the upgrade risk to DCIS - IC after diagnosis on VAB was: ADH 17,3%- 2,7%, FEA 11,7%- 5,9%, LN 0,0% - 4,3%, PL 10,4% - 2,1%. We found no upgrade for RS. (Table 1). Conclusions: In a series of B3 lesions with a histological follow-up, we notice that overall upgrade risk is higher for lesions detected on CNB than on VAB: 19,0% vs. 14,9%. The majority of lesions showed histological agreement between initial B3 diagnosis and histological follow-up: 57,9% after CNB and 53,5% after VAB. More investigation is needed to make a proper risk assessment as to which B3 lesions can be followed with regular surveillance. Also, further prospective research is needed to get a better understanding of associated risk factors for upgrade, upgrade risk and lifetime risk of developing breast cancer after diagnosis of a B3 lesion. Table 1: Upgrade risk per B3 subtype after diagnosis on CNB or VABSubtypeNumberCNB followed by VAB or WEDCISICTotal Upgrade riskCNBNumberVAB followed by WEDCISICUpgrade riskVAB followed by WEADH10517,1%12,4%29,5%11017,3%2,7%20,0%FEA3821,1%18,4%39,5%5111,7%5,9%17,6%LN3718,9%21,6%40,5%460%4,3%4,3%PL3367,2%3,2%10,4%4810,4%2,1%12,5%RS3514,3%11,4%25,7%60%0%0%Total551261Upgrade105/55119,0%39/26114,9%B3 lesion319/55157,9%138/26152,9%No B3 lesion120/55121,8%82/26131,4%Result not available7/5511,3%2/2610,8% Citation Format: Nynke Willers, Patrick Neven, Giuseppe Floris, Cecile Colpaert, Eva Oldenburger, Sileny Han, Chantal Van Ongeval, Ann Smeets, Francois Duhoux, Hans Wildiers, Petra Denolf, Isabel De Brabander, Nancy Van Damme, Harlinde De Schutter. The upgrade risk to (pre-)invasive breast cancer for B3 lesions diagnosed on core needle or vacuum assisted biopsy. A Belgian retrospective study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-52.

Positive predictive value for malignancy of uncertain malignant potential (B3) breast lesions diagnosed on vacuum-assisted biopsy (VAB): is surgical excision still recommended?

Breast lesions classified as of "uncertain malignant potential" represent a heterogeneous group of abnormalities with an increased risk of associated malignancy. Clinical management of B3 lesions diagnosed on vacuum-assisted breast biopsy (VABB) is still challenging: surgical excision is no longer the only available treatment and VABB may be sufficient for therapeutic excision. The aim of the present study is to evaluate the positive predictive value (PPV) for malignancy in B3 lesions that underwent surgical excision, identifying possible upgrading predictive factors and characterizing the malignant lesions eventually diagnosed. These results are compared with a subset of patients with B3 lesions who underwent follow-up. A total of 1250 VABBs were performed between January 2006 and December 2017 at our center. In total, 150 B3 cases were diagnosed and 68 of them underwent surgical excision. VABB findings were correlated with excision histology. A PPV for malignancy for each B3 subtype was derived. The overall PPV rate was 28%, with the highest upgrade rate for atypical ductal hyperplasia (41%), followed by classical lobular neoplasia (29%) and flat epithelial atypia (11%). Only two cases of carcinoma were detected in the follow-up cohort, both associated with atypical ductal hyperplasia at VABB. Open surgery is recommended in case of atypical ductal hyperplasia while, for other B3 lesions, excision with VABB only may be an acceptable alternative if radio-pathological correlation is assessed, if all microcalcifications have been removed by VABB, and if the lesion lacks high-risk cytological features. • Surgical treatment is strongly recommended in case of ADH, while the upgrade rate in case of pure FEA, especially following complete microcalcification removal by VABB, may be sufficiently low to advice surveillance as a management strategy. • The use of 11-G- or 8-G-needle VABB, resulting in possible complete diagnostic excision of the lesion, can be an acceptable alternative in case of RS, considering open surgery only for selected high-risk patients. • LN management is more controversial: surgical excision may be recommended following classical LN diagnosis on breast biopsy if an additional B3 lesion is concurrently detected while in the presence of isolated LN with adequate radiological-pathological correlation follow-up alone could be an acceptable option.

Prevalence of autoimmune diseases in thymic epithelial tumors (TET) insights from RYTHMIC.

9073 Background: TET are associated with autoimmune disorders (AID) in up to 30% of patients (pts). However, there have been wide variations in the reported prevalence of AID in TET pts in small single-center series. RYTHMIC (Réseau tumeurs THYMiques et Cancer) is a French network mandated to systematically discuss every case of TET. We aimed to describe the prevalence of AID in a large French population. Methods: RYTHMIC database, hosted by IFCT (Intergroupe Francophone de Cancérologie Thoracique), prospectively includes all consecutive pts with a diagnosis of TET discussed in French national or regional tumor boards. We analyzed epidemiologic, clinical and pathological characteristics of pts with TET’s related AID. Results: From January 2012 to December 2019, 2909 pts were included in the database. The mean age at diagnosis of TET was 54 and 52% were male. In the overall population, Masaoka Koga stages were well balanced with 12.6% (n = 187) stage I, 8.8% (n = 131) stage IIa, 8.4% (n = 124) stage IIb, 11.1% (n = 164) stage III and 8.5% (n = 125) stage IV. There were 364 (12.5%) events of AID in 302 pts. 62 pts (17%) had more than 1 AID. Among the events, 236 were myasthenia gravis (MG) (64.8%), 19 Hypo-gammaglobulinemia syndrome (5.2%), 15 pure red cell aplasia (4.1%), 18 thyroiditis (4.9%) and 16 systemic erythematous lupus (4.4%). Diagnosis of AID was mostly done at tumor diagnosis (n = 239, 65.7%) but some patient had AID diagnosed before diagnosis (n = 67, 18.4%) or during follow up (n = 32, 8.8%). Among pts presenting AID, B2 was the most common subtype (n = 133, 36.5%). The incidence of AID per subtype was as follow: A (n = 10/81, 12.3%), AB (n = 48/225, 21.3%), B1 (n = 35/130, 26.9%), B2 (n = 133/295, 45.0%), B3 (n = 46/113, 40.7%), thymic carcinoma (n = 16/275, 5.8%). Conclusions: The prevalence of AID in pts with TET was 12.5%, > 40% in B2 and B3 subtypes. Diagnosis of AID can be delayed compared to the diagnosis of TET. Immunotherapy indication should be carefully assessed in pts with TET other than thymic carcinoma.

Genetic characteristics of coxsackievirus A16 isolated in Shenzhen from 2016 to 2017

Objective To investigate the genetic characteristics of VP1 genes carried by coxsackievirus A16 strains isolated from cases of hand foot and mouth disease (HFMD) in Shenzhen during 2016 to 2017. Methods Fecal and anal swab specimens were collected from patients with mild HFMD in four sentinel hospitals and the Institute of Pathogen Biology, Shenzhen Center for Disease Control and Prevention, China during 2016 to 2017. All specimens were tested for CVA16 viral RNA using real-time RT-PCR. The VP1 genes of 51 randomly selected CVA16 strains were amplified by RT-PCR and then sequenced using TaKaRa Biomedical Technology (Dalian). Bioinformatics software, including Mega6.02, BioEdit and DNAStar, was used for comparison and analysis of the VP1 genes. Results CVA16 strains in Shenzhen during 2016 to 2017 mainly belonged to B1a and B1b subtypes as well as an emerging subtype B3. The epidemic of B1b subtype was found in both 2016 (28 strains) and 2017 (19 strains), while the B1a subtype (two strains) was only detected in 2017. Two B3 subtype strains were detected in 2017. The strains of B1b subtype were closely related to the strains isolated in Shanghai (JQ314149), Wenzhou (KP289416) and Beijing (KU254598), while the B1a subtype strains were closely related to the strains isolated in Kunming (JQ316639) and Tailand (GQ184139). The B3 subtype strain was an emerging CVA16 epidemic strain in mainland China. Further comparison of the CVA16 epidemic strains in Shenzhen area during 2016 to 2017 with the CVA16 strains causing severe neurological symptoms showed that two amino acid mutations (S14N and M23L) were found in VP1 protein. Conclusions The epidemic strains of CVA16 were B1b subtype in Shenzhen area in 2016. However, B1a, B1b and the emerging B3 subtype strains were prevalent in 2017. Compared with the CVA16 strains causing severe neurological symptoms, the CVA16 strains circulating in Shenzhen during 2016 to 2017 carried two amino acid mutations inVP1 protein. Key words: Coxsackievirus A16; Hand, foot and mouth disease; Phylogenetic analysis

B3 lesions of the breast; is watchful waiting an option?

Introduction: Breast lesions of uncertain malignant potential (B3) represent a heterogeneous group of pathologies with a 10 - 30% overall risk of malignancy diagnosis after surgical excision. Malignancy risk varies depending on B3 subtypes. With such heterogeneity, ideal management of patients with B3 lesions is unclear.

Proteomic Signatures of Thymomas.

Based on the histological features and outcome, the current WHO classification separates thymomas into A, AB, B1, B2 and B3 subtypes. It is hypothesized that the type A thymomas are derived from the thymic medulla while the type B thymomas are derived from the cortex. Due to occasional histological overlap between the tumor subtypes creating difficulties in their separation, the aim of this study was to provide their proteomic characterization and identify potential immunohistochemical markers aiding in tissue diagnosis. Pair-wise comparison of neoplastic and normal thymus by liquid chromatography tandem mass spectrometry (LC-MS/MS) of formalin fixed paraffin embedded tissue revealed 61 proteins differentially expressed in thymomas compared to normal tissue. Hierarchical clustering showed distinct segregation of subtypes AB, B1 and B2 from that of A and B3. Most notably, desmoyokin, a protein that is encoded by the AHNAK gene, was associated with type A thymomas and medulla of normal thymus, by LC-MS/MS and immunohistochemistry. In this global proteomic characterization of the thymoma, several proteins unique to different thymic compartments and thymoma subtypes were identified. Among differentially expressed proteins, desmoyokin is a marker specific for thymic medulla and is potentially promising immunohistochemical marker in separation of type A and B3 thymomas.

Molecular characterization of measles viruses that circulated in Cameroon between 2010 and 2011.

BackgroundMeasles virus (MeV) is monotypic, but genetic variation in the hemagglutinin H and nucleoprotein N genes can be analyzed by molecular epidemiologic techniques and used to study virus transmission patterns. The World Health Organization currently recognizes 8 clades (A-H) within which are 24 genotypes of MeV and one provisional genotype, d11. Genotype B3 is clearly the endemic genotype in most of African continent where it is widely distributed. We provide an update on the molecular characterization of wild-type MeVs that circulated in Cameroon between 2010 and 2011.FindingsViral RNA was extracted directly from samples obtained from clinically diagnosed measles patients using QIAamp viral RNA Mini Kit. Reverse transcription and PCR amplification of 634 nucleotides of the N gene was performed using the SuperScript™ III One-Step. Sequence analysis of 450 of the 634 nucleotides using Clustal X 2.0 program for multiple alignments and Mega version 5 for phylogenic analysis indicated that all the viruses belonged to genotype B3 with two distinct clusters. Twenty three (77%) belonged to subgroup B3.1 and the other 7 (23%) belonged to B3.3 a recently described subtype. Circulation of cluster 3 was detected in the Far-North Region (5/7) particularly along the Chad-Cameroon border in 2010 and later in Yaounde (2/7 in Biyem-assi Health District) the capital city of Cameroon in 2011.ConclusionThis study highlights the endemic circulation in Cameroon of MeV B3 subtype 1, which probably has its source in the neighboring Nigeria, and the presence of the new subtype B3.3, suggesting a possible importation from Northern Africa where it was first described between 2008 and 2009.

Diagnosis and subclassification of thymoma by minimally invasive fine needle aspiration directed by endobronchial ultrasound: a review and discussion of four cases

Thymomas have been classified by the World Health Organisation (WHO) into six groups, based on the morphology of epithelial cells and the ratio between epithelial cells and lymphocytes within the tumour. Among 1458 consecutive cases of endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) performed in a tertiary referral centre between February 2008 and February 2012, we have encountered four cases of thymic neoplasms. We discuss the cytomorphological features of three cases of type B thymoma (one each of B1, B2 and B3 subtypes) and one case of thymic carcinoma diagnosed on EBUS-TBNA using cell blocks, immunocytochemistry and flow cytometry which allowed preoperative chemotherapy to be carried out in two cases, diagnosis to be made after unsatisfactory surgical mediastinoscopy in the third and diagnosis of lymph node metastasis of the thymic carcinoma in the fourth. The differential diagnosis and criteria for subclassification of thymomas are discussed; although subclassification of these cases was possible in these cases, and tumours other than thymoma excluded, additional cases would be necessary to assess the potential accuracy of EBUS-TBNA. These, to the best of our knowledge, represent the first cases of thymoma that were diagnosed and subclassified according to WHO criteria using multimodality evaluation of EBUS-derived cytological aspirates.

Positive predictive value for malignancy on surgical excision of breast lesions of uncertain malignant potential (B3) diagnosed by stereotactic vacuum-assisted needle core biopsy (VANCB): A large multi-institutional study in Italy

Percutaneous core biopsy (CB) has been introduced to increase the ability of accurately diagnosing breast malignancies without the need of resorting to surgery. Compared to conventional automated 14 gauge needle core biopsy (NCB), vacuum-assisted needle core biopsy (VANCB) allows obtaining larger specimens and has recognized advantages particularly when the radiological pattern is represented by microcalcifications. Regardless of technical improvements, a small percentage of percutaneous CBs performed to detect breast lesions are still classified, according to European and UK guidelines, in the borderline B3 category, including a group of heterogeneous lesions with uncertain malignant potential. We aimed to assess the prevalence and positive predictive values (PPV) on surgical excision (SE) of B3 category (overall and by sub-categories) in a large series of non-palpable breast lesions assessed through VANCB, also comparison with published data on CB. Overall, 26,165 consecutive stereotactic VANCB were identified in 22 Italian centres: 3107 (11.9%) were classified as B3, of which 1644 (54.2%) proceeded to SE to establish a definitive histological diagnosis of breast pathology. Due to a high proportion of microcalcifications as main radiological pattern, the overall PPV was 21.2% (range 10.6%–27.3% for different B3 subtypes), somewhat lower than the average value (24.5%) from published studies (range 9.9%–35.1%). Our study, to date the largest series of B3 with definitive histological assessment on SE, suggests that B3 lesions should be referred for SE even if VANCB is more accurate than NCB in the diagnostic process of non-palpable, sonographically invisible breast lesions.

Flat epithelial atypia is a common subtype of B3 breast lesions and is associated with noninvasive cancer but not with invasive cancer in final excision histology

The biological behavior and the optimal management of benign breast lesions with uncertain malignant potential, the so-called B3 lesions, found in breast needle core biopsies is still under debate. We addressed this study to compare histologic findings in B3 needle core biopsies with final excision specimens to determine associated rates of malignancy. Consecutive needle core biopsies were performed in a 3-year period (January 1, 2006-December 31, 2008). Biopsies were image-guided (31 by ultrasound, 85 stereotactic vacuum-assisted, 6 unknown) for evaluation of breast abnormalities. We reviewed 122 needle core biopsies with B3 lesions of 91 symptomatic patients and 31 screen-detected women and compared the B3 histologic subtypes with the final excision histology. A total of 1845 needle core biopsies were performed and B3 lesions comprised 6.6% of all B categories. The most common histologic subtype in biopsies was flat epithelia atypia in 35.2%, followed by papillary lesions in 21% and atypical ductal hyperplasia in 20%. Reports on excision specimens were available in 66% (81 patients). Final excision histology was benign in 73 (90.2%) and malignant in 8 (9.8%) patients (2 invasive cancer, 6 ductal carcinoma in situ). Of all B3 subtypes, atypical ductal hyperplasia and flat epithelial atypia were associated with malignancy, whereas only atypical ductal hyperplasia was accompanied by invasive cancer. Of all lesions, flat epithelial atypia was most frequently found in excision specimens (18%). In our study, flat epithelial atypia and atypical ductal hyperplasia are common lesions of the B3 category in needle core biopsies of the breast. Both lesions are associated with malignancy, whereas only atypical ductal hyperplasia was related to invasive cancer. We conclude that an excision biopsy after diagnosis of flat epithelial atypia is recommended depending on clinical and radiologic findings.

A Dispermic Chimera with Mixed Field Blood Group B and Mosaic 46,XY/47,XYY Karyotype

Chimerism in humans is a rare phenomenon often initially identified in the resolution of an ABO blood type discrepancy. We report a dispermic chimera who presented with mixed field in his B antigen typing that might have been mistaken for the B3 subtype. The propositus is a healthy Korean male blood donor. Neither his clinical history nor initial molecular investigation of his ABO gene explained his mixed field agglutination with murine anti-B. Chimerism was suspected, and 9 short tandem repeat (STR) loci were analyzed on DNA extracted from blood, buccal swabs, and hair from this donor and on DNA isolated from peripheral blood lymphocytes from his parents. The propositus' red blood cells demonstrated mixed field agglutination with anti-B. Exon 6 and 7 and flanking intronic regions of his ABO gene were sequenced and revealed an O01/O02 genotype. B allele haplotype-specific PCR, along with exon 6 and 7 cloning and sequencing demonstrated a third ABO allele, B101. Four STR loci demonstrated a pattern consistent with a double paternal chromosome contribution in the propositus, thus confirming chimerism. His karyotype revealed a mosaic pattern: 32/50 metaphases were 46,XY and 18/50 metaphases demonstrated 47,XYY.

Molecular cloning, functional expression and pharmacological characterization of a human bradykinin B2 receptor gene

The gene encoding a putative G protein-coupled receptor (HG10) was cloned from human genomic DNA by low stringency PCR and found to be homologous to the recently described rat bradykinin B2 receptor (1). The receptor was expressed in xenopus oocytes and stably transfected CHO cell lines. Binding studies demonstrated that HG10 encodes a high affinity BK receptor with an apparent Kd of 150 pM. Displacement by BK agonists and antagonists allowed the characterization of the receptor as a B2 subtype. Functional coupling to the Ca 2+-phosphatidylinositol cascade was demonstrated in transfected CHO cells where inositol phosphates accumulation and intracellular calcium concentration were elevated in response to BK stimulation. The agonistic and antagonistic properties of BK analogs do not match strictly the pharmacological profile described for the rat or guinea pig B2 receptor subtypes or the putative B3 subtype (2). This discrepancy is attributed either to species variability or to differences in the coupling efficiency of receptors to the transduction cascade in different cell types. From our results, the existence of B3 receptors and of B2 subtypes appears questionable.

Ultrastructural features of functionally identified primary afferent neurons with C (unmyelinated) fibers of the guinea pig: classification of dorsal root ganglion cell type with reference to sensory modality.

Intracellular labeling of neurons permitted a direct correlation of neuronal profiles with sensory modality of cutaneous receptors. In the guinea pig, 47 neurons in the dorsal root ganglion (displaying C-fiber conduction velocities) were labeled with horseradish peroxidase (HRP) by iontophoresis after determining the sensory modality. Receptive field were explored with systematic "natural" stimuli. Cell areas of all C-fiber units were measured by tracing the cellular contour in light microscopy. The mean cellular diameter calculated from cell areas was 21.8 microns in the second cervical ganglion of the guinea pig. Mean cell diameter for high-threshold mechanoreceptors was 20.9 microns, 24.6 microns for polymodal nociceptors, and 25.7 microns for mechanical-cold nociceptors. Electron microscopic observations showed that all labeled neurons of C-fiber units had profiles of small, dark type-B neurons. Neurons representative of each sensory modality exhibited different cell features, each belonging to a distinct subtype of small B neurons. High-threshold mechanoreceptor and mechanical-cold nociceptor displayed a peripheral lamellar arrangement of cisternae of endoplasmic reticulum (ER), corresponding to the B1 subtype. Polymodal nociceptor units were characteristically of the B2 subtype, in which stacks of long and short cisternae of ER were distributed randomly throughout the cytoplasm, and the arrangement of Golgi bodies varied among these cells. Cooling receptors displayed poorly developed, flattened cisternae of ER and numerous vesicles, typical of the B3 subtype. These results imply that all C-fiber cells belong to the small B-type cell category and that the ultrastructural features of the neuron in the dorsal root ganglion may reflect the sensory modality of the receptive field.