Single-Cell RNA Sequencing for the Detection of Clonotypic V(D)J Rearrangements in Multiple Myeloma.

Multiple myeloma (MM) has a highly heterogeneous genetic background, which complicates its molecular tracking over time. Nevertheless, each MM patient's malignant plasma cells (PCs) share unique V(D)J rearranged sequences at immunoglobulin loci, which represent ideal disease biomarkers. Because the tumor-specific V(D)J sequence is highly expressed in bulk RNA in MM patients, we wondered whether it can be identified by single-cell RNA sequencing (scRNA-seq). To this end we analyzed CD138+ cells purified from bone marrow aspirates of 19 samples with PC dyscrasias by both a standard method based on bulk DNA and by an implementation of the standard 10x Genomics protocol to detect expressed V(D)J sequences. A dominant clonotype was easily identified in each sample, accounting on average for 83.65% of V(D)J-rearranged cells. Compared with standard methods, scRNA-seq analysis proved highly concordant and even more effective in identifying clonal productive rearrangements, by-passing limitations related to the misannealing of consensus primers in hypermutated regions. We next validated its accuracy to track 5 clonal cells with absolute sensitivity in a virtual sample containing 3180 polyclonal cells. This shows that single-cell V(D)J analysis may be used to find rare clonal cells, laying the foundations for functional single-cell dissection of minimal residual disease.

Inference of genomic lesions from single-cell RNA-seq in myeloma improves functional intraclonal and interclonal analysis.

ABSTRACT: Smoldering multiple myeloma (SMM) is an asymptomatic plasma cell (PC) neoplasm that may evolve with variable frequency into multiple myeloma (MM). SMM is initiated by chromosomal translocations involving the immunoglobulin heavy-chain locus or by hyperdiploidy and evolves through acquisition of additional genetic lesions. In this scenario, we aimed at establishing a reliable analysis pipeline to infer genomic lesions from transcriptomic analysis, by combining single-cell RNA sequencing (scRNA-seq) with B-cell receptor sequencing and copy number abnormality (CNA) analysis to identify clonal PCs at the genetic level along their specific transcriptional landscape. We profiled 20 465 bone marrow PCs derived from 5 patients with SMM/MM and unbiasedly identified clonal and polyclonal PCs. Hyperdiploidy, t(11;14), and t(6;14) were identified at the scRNA level by analysis of chimeric reads. Subclone functional analysis was improved by combining transcriptome with CNA analysis. As examples, we illustrate the different functional properties of a light-chain escape subclone in SMM and of different B-cell and PC subclones in a patient affected by Wäldenstrom macroglobulinemia and SMM. Overall, our data provide a proof of principle for inference of clinically relevant genotypic data from scRNA-seq, which in turn will refine functional annotation of the clonal architecture of PC dyscrasias.

MGUS and clonal hematopoiesis show unrelated clinical and biological trajectories in an older population cohort.

Monoclonal gammopathy of undetermined significance (MGUS) and clonal hematopoiesis (CH) are 2 preclinical clonal expansions of hematopoietic cells whose prevalence rises with age, reaching almost 10% in people of aged 70 years and older. The increased risk of myeloid malignancies in patients with myeloma is well defined, and the study of the association between CH and MGUS could help explain this phenomenon. Here, we analyzed a fully clinically annotated dataset of 777 older subjects (median age, 91 years) previously screened for prevalence of CH. The prevalence of MGUS and CH was 9.6% and 17.3%, respectively. We detected CH in 9.7% of the patients with MGUS and MGUS in 5.5% of the patients with CH. We did not find a significant correlation between the presence of MGUS and CH. Furthermore, the 2 conditions showed a differential association with clinical and laboratory covariates, suggesting that MGUS and CH may represent age-associated unrelated clonal drifts of hematopoietic cells. Confirmatory studies are needed to assess the relevance of CH in plasma cell disorders. This trial was registered at www.clinicaltrials.gov as #NCT03907553.

Correlates of HCV seropositivity among familial contacts of HCV positive patients.

BACKGROUND: Determinants of intrafamilial HCV transmission are still being debated. The aim of this study is to investigate the correlates of HCV seropositivity among familial contacts of HCV positive patients in Italy. METHODS: A cross-sectional study was conducted with 175 HCV positive patients (index cases), recruited from Policlinico Gemelli in Rome as well as other hospitals in Central Italy between 1995 and 2000 (40% female, mean age 57 +/- 15.2 years), and 259 familial contacts. Differences in proportions of qualitative variables were tested with non-parametric tests (chi2, Yates correction, Fisher exact test), and a p value < 0.05 was considered significant. A multivariate analysis was conducted using logistic regression in order to verify which variables statistically have an influence on HCV positivity in contact individuals. RESULTS: Seropositivity for HCV was found in 8.9% of the contacts. From the univariate analysis, risk factors significantly associated to HCV positivity in the contacts were: intravenous drug addiction (p = 0.004) and intercourse with drug addicts (p = 0.005). The only variables associated significantly and independently to HCV seropositivity in patients' contacts were intercourse with drug addicts (OR = 19.28; 95% CI: 2.01 - 184.94), the retirement status from work (OR = 3.76; 95% CI: 1.17 - 11.98), the time of the relationship (OR = 1.06; 95% CI: 1.00 - 1.11) and tattoos (OR = 7.68; 95% CI: 1.00 - 60.20). CONCLUSION: The present study confirms that having intercourse with a drug addict is the most significant risk factor for intrafamilial HCV transmission. The association with retirement status from work could be related to both a long-term relationship with an index case and past exposure to common risk factors.

Comparação entre os efeitos da associação gelatina-resorcina-formaldeído e do n-butil-2-cianoacrilato na síntese do parênquima hepático de coelhos / Comparison between the effects of the compound gelatin-resorsin-formaldeide and the n-butil-2-cianoacrilate in hepatectomy in rabbits

Considerando-se as particularidades anatômicas e funcionais do fígado e a dificuldade da realização de procedimentos cirúrgicos eficientes quanto a hemostasia, estudou-se comparativamente os efeitos de dois adesivos cirúrgicos, gelatina-resorsina-formaldeído e n-butil-2-cianoacrilato, como métodos auxiliares na síntese do órgão. A metodologia utilizada determinou rigoroso acompanhamento clínico dos animais estudados, assim como realização de análises macro e microscópica de feridas cirúrgicas experimentalmente induzidas, quanto à evolução do processo cicatricial. Os resultados obtidos demonstraram hemostasia rápida e eficiente em ambos grupos, e alterações celulares e teciduais mais significativas no grupo tratado com n-butil-2-cianoacrilato, quando comparadas àquelas observadas nos grupos controle e gelatina-resorcina-formaldeído. Em relação à proposição estudada, o composto gelatina-resorsina-formaldeído apresentou-se como método alternativo de escolha a ser utilizado nos casos cujas suturas aplicadas isoladamente não proporcionem hemostasia satisfatória.(AU)

Treatment of ear haematoma of the dog with Triamcinolone acetonide / Tratamento do oto-hematoma do cão com o emprego do acetonil-traimcinolona

The authors reported a now method of trating so called hoematoma affecting dogs ear, using in all cases an injection of triamcinolone acetonide (10 mg/ml), and all the fluid contained was removed prior the injection. After looking over the literature concerning and discussing the results obtained, the authors were able to conclude: The value of treatment with the synthetic corticosteroid injection has definitively been established and has given satisfactory results in at least 83,3 percent of the cases treated; 52 percent of these, have hand only one injection, 40 percent two and 8 percent three injections. In all cases the injections were done every 7 days. They didn"t verify the thickness of the ear in 52 percent of the cases treated.

O artigo apresenta resumo em inglês.

Treatment of ear haematoma of the dog with Triamcinolone acetonide / Tratamento do oto-hematoma do cão com o emprego do acetonil-traimcinolona

The authors reported a now method of trating so called hoematoma affecting dogs ear, using in all cases an injection of triamcinolone acetonide (10 mg/ml), and all the fluid contained was removed prior the injection. After looking over the literature concerning and discussing the results obtained, the authors were able to conclude: The value of treatment with the synthetic corticosteroid injection has definitively been established and has given satisfactory results in at least 83,3 percent of the cases treated; 52 percent of these, have hand only one injection, 40 percent two and 8 percent three injections. In all cases the injections were done every 7 days. They didn"t verify the thickness of the ear in 52 percent of the cases treated.

O artigo apresenta resumo em inglês.

General anesthesia in the dog. II. Different doses of pentobarbital sodium with previous injection of xylazine / Anestesia geral no cão. II. Variação da quantidade de pentobarbital sódico em animais pré-medicados com cloridrato de xilazina

Anesthesia by endovenous injection of 1,01 to 1,50mg/kg of xylazine followed by administration of different doses of pentobarbital sodium were studied in 25 dogs. It was observed different times of animal surgical anesthesia as so in the necessary times to recover and those verified in 5 control dogs. Statistical analysis revealed significant differences (P .05) among control and treated groups.

São estudados em 25 animais, a anestesia determinada pela aplicação endovenosa de 1,01 a 1,50 miligramas de cloridrato dexilazina a para cada quilograma de peso corpóreo seguida da administração de doses variáveis e crescentes de pentobarbital sódico. É observada variação dos tempos de anestesia e necessários para o retorno que, comparados aos resultados de um lote controle, mostram diferenças significativas ao nível de rejeição de 0,05.

General anesthesia in the dog. I. Pentobarbital sodium with previous injection of xylazine / Anestesia geral no cão. I. Utilização do pentobarbital sódico com administração prévia de cloridrato de xilazina

Effects of endovenous injection of xylazine 2% followed by administration of pentobarbital sodium 3% were studied in30 dogs. A comparative study was made between the results observed in 5 dogs anesthetised only with pentobarbital sodium and those verified in 5 experimental groups of 5 dogs each. It was observed a reduction in the consumption of barbituric, at average times of animal surgical anesthesia, as so in the necessary time to recover. Arterial pulse and respiratory frequencies decresaed immediately after drugs injections. Statistical analysis revealed significant difference (P .05) only in the rectal temperature of control animals.

São estudados em 30 cães, os resultados obtidos com a aplicação endovenosa de cloridrato de xilazina a 2% seguida da administração de pentobarbital sódico a 3%. São comparados os resultados com os obtidos em um lote de 5 animais anestesiados exclusivamente com pentobarbital sódico e observados, nos lotes experimentais, redução da quantidade do barbitúrico consumido e dos tempos médios de duração da fase de anestesia cirúrgica e necessário para o retorno. As frequências de pulso e respiratória mostraram-se diminuídas logo após a aplicação das 2 drogas. No tocante a temperatura retal apenas os animais do lote controle apresentaram diferenças significativas ao nível de 0,05.

Experimental rumen fistula in bovine / Fístula experimental do rúmen em bovinos

We have studied the surgical technique of a cannulated experimental fistula by suture (fixation) of the rumen to the abdominal muscles in cattle. Rumenostomy in standing position with infiltrative anaesthesia and only one surgical procedure was performed without postoperative complications in 10 animals. It was observed a good adaptation of the cannula to the fistula, with development of adherence between the rumen and the abdominal wall and continuity of its mucosa with the skin.

Os autores relatam a realização de fistula experimental canulada do rúmen em 10 animais da espécie bovina. A técnica da rumenostomia foi efetuada em apenas um ato operatório, com os animais em estação, utilizando-se a anestesia local infiltrativa e com fixação do rúmen na parede muscular do abdômen. O procedimento mostrou-se satisfatório, com perfeita adaptação da cânula, formação de aderência completa do rúmen e parede abdominal e continuidade de sua mucosa com o revestimento cutâneo, sem complicações pós-operatórias.

Surgical correction of faulty ears with polypropylene mesh / Correção cirúrgica de defeitos do pavilhão auricular. Emprego de malha de polipropileno

A surgical technique to correct faulty ears by polyprolylene mesh implantation was studied in 8 dogs. Satisfactory results were obtained in 7 dogs which showed erectness and proper movements of the ears. No retraction or thickning at the surgical site were observed.

Foi estudada, em 8 animais da espécie canina, uma técnica operatória para correção de defeitos do pavilhão auricular, mediante  implantação de malha de polipropileno. Os resultados foram satisfatórios em 7 cães, que mostraram posição ereta e movimentação adequada das orelhas e ausência de retrações ou espessamento dos pavilhões auriculares.  

Ear cropping: measurements and weight characteristics of the auricular pieces / Conchotomia: características métricas e ponderal dos retalhos auriculares

The measurements and weight characteristics of the excised auricular pieces at standardized ear cropping were observed in 24 Doberman Pincher dogs. Good aesthetical aspects were observed in different ages.

Os autores determinaram, em 24 cães da raça Doberman, as características métricas e ponderal dos retalhos auriculares excisados na operação de conchotomia realizada mediante manobras padronizadas. Não foram observadas diferenças evidentes ou estatisticamente significativas nas diferentes idades estudadas e os animais mostraram resultados estéticos favoráveis.

General anesthesia in the dog. I. Pentobarbital sodium with previous injection of xylazine / Anestesia geral no cão. I. Utilização do pentobarbital sódico com administração prévia de cloridrato de xilazina

Effects of endovenous injection of xylazine 2% followed by administration of pentobarbital sodium 3% were studied in30 dogs. A comparative study was made between the results observed in 5 dogs anesthetised only with pentobarbital sodium and those verified in 5 experimental groups of 5 dogs each. It was observed a reduction in the consumption of barbituric, at average times of animal surgical anesthesia, as so in the necessary time to recover. Arterial pulse and respiratory frequencies decresaed immediately after drugs injections. Statistical analysis revealed significant difference (P .05) only in the rectal temperature of control animals.

São estudados em 30 cães, os resultados obtidos com a aplicação endovenosa de cloridrato de xilazina a 2% seguida da administração de pentobarbital sódico a 3%. São comparados os resultados com os obtidos em um lote de 5 animais anestesiados exclusivamente com pentobarbital sódico e observados, nos lotes experimentais, redução da quantidade do barbitúrico consumido e dos tempos médios de duração da fase de anestesia cirúrgica e necessário para o retorno. As frequências de pulso e respiratória mostraram-se diminuídas logo após a aplicação das 2 drogas. No tocante a temperatura retal apenas os animais do lote controle apresentaram diferenças significativas ao nível de 0,05.

General anesthesia in the dog. II. Different doses of pentobarbital sodium with previous injection of xylazine / Anestesia geral no cão. II. Variação da quantidade de pentobarbital sódico em animais pré-medicados com cloridrato de xilazina

Anesthesia by endovenous injection of 1,01 to 1,50mg/kg of xylazine followed by administration of different doses of pentobarbital sodium were studied in 25 dogs. It was observed different times of animal surgical anesthesia as so in the necessary times to recover and those verified in 5 control dogs. Statistical analysis revealed significant differences (P .05) among control and treated groups.

São estudados em 25 animais, a anestesia determinada pela aplicação endovenosa de 1,01 a 1,50 miligramas de cloridrato dexilazina a para cada quilograma de peso corpóreo seguida da administração de doses variáveis e crescentes de pentobarbital sódico. É observada variação dos tempos de anestesia e necessários para o retorno que, comparados aos resultados de um lote controle, mostram diferenças significativas ao nível de rejeição de 0,05.

Ear cropping: measurements and weight characteristics of the auricular pieces / Conchotomia: características métricas e ponderal dos retalhos auriculares

The measurements and weight characteristics of the excised auricular pieces at standardized ear cropping were observed in 24 Doberman Pincher dogs. Good aesthetical aspects were observed in different ages.

Os autores determinaram, em 24 cães da raça Doberman, as características métricas e ponderal dos retalhos auriculares excisados na operação de conchotomia realizada mediante manobras padronizadas. Não foram observadas diferenças evidentes ou estatisticamente significativas nas diferentes idades estudadas e os animais mostraram resultados estéticos favoráveis.

Surgical correction of faulty ears with polypropylene mesh / Correção cirúrgica de defeitos do pavilhão auricular. Emprego de malha de polipropileno

A surgical technique to correct faulty ears by polyprolylene mesh implantation was studied in 8 dogs. Satisfactory results were obtained in 7 dogs which showed erectness and proper movements of the ears. No retraction or thickning at the surgical site were observed.

Foi estudada, em 8 animais da espécie canina, uma técnica operatória para correção de defeitos do pavilhão auricular, mediante  implantação de malha de polipropileno. Os resultados foram satisfatórios em 7 cães, que mostraram posição ereta e movimentação adequada das orelhas e ausência de retrações ou espessamento dos pavilhões auriculares.

Experimental rumen fistula in bovine / Fístula experimental do rúmen em bovinos

We have studied the surgical technique of a cannulated experimental fistula by suture (fixation) of the rumen to the abdominal muscles in cattle. Rumenostomy in standing position with infiltrative anaesthesia and only one surgical procedure was performed without postoperative complications in 10 animals. It was observed a good adaptation of the cannula to the fistula, with development of adherence between the rumen and the abdominal wall and continuity of its mucosa with the skin.

Os autores relatam a realização de fistula experimental canulada do rúmen em 10 animais da espécie bovina. A técnica da rumenostomia foi efetuada em apenas um ato operatório, com os animais em estação, utilizando-se a anestesia local infiltrativa e com fixação do rúmen na parede muscular do abdômen. O procedimento mostrou-se satisfatório, com perfeita adaptação da cânula, formação de aderência completa do rúmen e parede abdominal e continuidade de sua mucosa com o revestimento cutâneo, sem complicações pós-operatórias.

Simplified surgical technique of ear cropping / Técnica operatória simplificada de conchotomia

A simplified surgical technique for ear cropping was studied in 120 dogs. The technique consisted of trimming of the ears after clamping, without the use of cauterization nor sutures. The wounds exhibited a faultless healing in a short time and few imperfections were observed at the ears. The cosmetic aspect was satisfatory.

Foi estudada, em 120 animais da espécie canina, técnica operatória de conchotomia com emprego de pinças limitativas e sem utilização de cauterização ou sutura. Os resultados mostraram-se satisfatórios e foram representados pela cicatrização perfeita e rápida das feridas operatórias, com baixa incidência de defeitos que comprometem a apresentação estética das orelhas.

Simplified surgical technique of ear cropping / Técnica operatória simplificada de conchotomia

A simplified surgical technique for ear cropping was studied in 120 dogs. The technique consisted of trimming of the ears after clamping, without the use of cauterization nor sutures. The wounds exhibited a faultless healing in a short time and few imperfections were observed at the ears. The cosmetic aspect was satisfatory.

Foi estudada, em 120 animais da espécie canina, técnica operatória de conchotomia com emprego de pinças limitativas e sem utilização de cauterização ou sutura. Os resultados mostraram-se satisfatórios e foram representados pela cicatrização perfeita e rápida das feridas operatórias, com baixa incidência de defeitos que comprometem a apresentação estética das orelhas.

General anesthesia in the dog. III Practical application of xylazine and pentobarbital sodium association / Anestesia geral no cão. III. Emprego da associação de cloridrato de xilazina e pentobarbital sódico

The behavior of 60 surgical patient dogs anesthetized endovenously with 1,01 - 1,50 mg/kg of xylazine followed by sodium pentobarbital as necessary for operation types, were studied. Groups of dogs were stablished based on both operation length and barbituric consumption. It was observed in 37 dogs (61,66%) a consumption of 5,09 mg/kg of pentobarbital/17,43 minutes of operation; 19 animals (31,66%) were operated with 10,27 mg/kg of pentobarbital in 30,26 minutes; 3 dogs (5,00%) used 15,02mg/kg in 48,33 minutes. In only one case it was necessary 18,92mg/kg for a period of 70 minutes.

Estudou-se o comportamento de 60 animais submetidos a tratamento cirúrgico durante anestesia fornecida pela injeção endovenosa de 1,01 a 1,50 mg/kg de cloridrato de xilazina e pentobarbital sódico administrado em quantidades diferentes e de acordo com a necessidade do ato operatório. Os resultados permitiram agrupar os animais segundo a duração do ato operatório e a quantidade de barbitúrico consumido 37 animais (61,66%) foram operados em 17,43 minutos em média, com a dose anestésica inicial, representada por 5,09 mg/kg de pentobarbital sódico; 19 animais (31,66%) necessitaram para 30,26 minutos, uma dose adicional, utilizando 10,27 mg/kg; 3 casos (5,00%) exigiram em 48,33 minutos, duas doses adicionais, totalizando 15,02 mg/kg e apenas um cão (1,66 %) recebeu três doses adicionais atingindo 18,92 mg/kg de barbitúrico para um ato cirúrgico realizado em 70,00 minutos.