RESUMO
PURPOSE: To determine the cellular composition of a product created with peripheral blood harvested after systemic mobilization with filgrastim and processed with one point-of-care blood concentrating system, i.e., a platelet-rich plasma (PRP) system. The second purpose was to compare mobilized platelet-rich plasma (M-PRP) with a concentrated bone marrow aspirate (cBMA) and a PRP created from the same subjects with the same PRP system. METHODS: Ten healthy volunteer subjects were recruited for collection and analysis of 3 tissue sources: non-treated peripheral blood, bone marrow aspirate, and filgrastim-mobilized peripheral blood, involving 4 doses of weight-based filgrastim. One point-of-care blood and bone marrow concentrating system was used to create 3 products: PRP, cBMA, and M-PRP. Automated hematologic analysis was performed on all products to quantify total red blood cells, white blood cells (WBCs), monocyte, platelet, and hematopoietic progenitor cell (HPC) concentrations. Flow cytometry was used to determine hematopoietic and mesenchymal progenitor cell populations. Lastly, concentrates were cultured and fibroblast colony-forming units (CFU-F) and morphology of adherent cells were evaluated. RESULTS: M-PRP contained a greater concentration of WBC (mean difference = 53.2 k/µL; P < .0001), monocytes (mean difference = 8.3 k/µL; P = .002), and a trend toward a greater concentration of HPC (mean difference = 200.5 /µL; P = .060) when compared with PRP. M-PRP contained a greater concentration of monocytes (mean difference = 5.5 k/µL; P = .017) and a trend toward a greater concentration of platelets (mean difference = 348 k/µL; P = .051) and HPC (mean difference = 193.4 /µL; P = .068) when compared with cBMA. M-PRP had a similar concentration of platelets to PRP (mean difference = 110 k/µL; P = .051) and PRP had a greater concentration than cBMA (mean difference = 458 k/µL; P = .003). cBMA remained the only product capable of producing CFU-Fs (446 ± 247 /mL) as neither the M-PRP nor PRP produced CFU-Fs. M-PRP produced colonies consistent with WBC. CONCLUSIONS: M-PRP, produced with filgrastim mobilized blood and a proprietary PRP system, contained more total WBCs, monocytes, platelets, and HPCs than cBMA and more WBCs, monocytes, and HPCs than PRP. CLINICAL RELEVANCE: Filgrastim mobilized PRP may be an alternative to cBMA for use as a point-of-care product for orthopaedic treatments.
Assuntos
Plaquetas/citologia , Células da Medula Óssea/citologia , Filgrastim/farmacologia , Células-Tronco Mesenquimais/citologia , Plasma Rico em Plaquetas , Adulto , Adesão Celular , Contagem de Células , Citometria de Fluxo , Humanos , Masculino , Adulto JovemRESUMO
The objective was to compare the mRNA expression of pro-inflammatory (TNF-α, IL-1ß, IFN-γ, IL-2, IL-12, IL-15) and anti-inflammatory (IL-4, IL-10, TGF-ß) cytokines, after experimental infection with low or high virulence noncytopathic (ncp) bovine viral diarrhea virus (BVDV). Thirty BVDV-naïve, beef calves were intranasally inoculated with low (LV; n=10, SD-1) or high (HV; n=10, 1373) virulence ncp BVDV or with BVDV-free cell culture medium (Control, n=10). Calves were euthanized on day 5 post-inoculation, and tracheo-bronchial lymph node and spleen samples were collected for mRNA expression through quantitative-RT-PCR. mRNA levels of pro-inflammatory (TNF-α, IL-1ß, IL-2, IFN-γ) and anti-inflammatory (IL-4 and IL-10) cytokines were up-regulated in tracheo-bronchial lymph nodes of HV, but not in LV, compared to the control group (P<0.05). IL-12 mRNA level was up-regulated in tracheo-bronchial lymph nodes of both LV and HV groups (P ≤ 0.05). A significant up-regulation of IL-15 mRNA was observed in tracheo-bronchial lymph nodes for LV calves (P<0.002), but not for HV calves. Experimental inoculation with BVDV-2 1373 stimulated significant mRNA expression of pro-inflammatory and anti-inflammatory cytokines. In contrast, inoculation with BVDV-1a SD-1 only resulted in up-regulation of IL-12 and IL-15 mRNA, which is associated with activation of macrophages and NK cells during innate immune response.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Citocinas/genética , Animais , Bovinos , Interleucina-1beta/genética , Interleucina-2/genética , RNA Mensageiro/análise , Fator de Necrose Tumoral alfa/genéticaRESUMO
Equine type 1 polysaccharide storage myopathy (PSSM1), a common glycogenosis associated with an R309H founder mutation in the glycogen synthase 1 gene (GYS1), shares pathological features with several human myopathies. In common with related human disorders, the pathogenesis remains unclear in particular, the marked phenotypic variability between affected animals. Given that affected animals accumulate glycogen and alpha-crystalline polysaccharide within their muscles, it is possible that physical disruption associated with the presence of this material could exacerbate the phenotype. The aim of this study was to compare the histopathological changes in horses with PSSM1, and specifically, to investigate the hypothesis that the severity of underlying pathology, (e.g. vacuolation and inclusion formation) would (1) be higher in homozygotes than heterozygotes and (2) correlate with clinical severity. Resting and post-exercise plasma creatine kinase (CK) and aspartate aminotransferase (AST) enzyme activity measurements and muscle pathology were assessed in matched cohorts of PSSM1 homozygotes, heterozygotes or control horses. Median (interquartile range (IR)) resting CK activities were 364 (332-764) U/L for homozygotes, 301 (222-377) U/L for heterozygotes and 260 (216-320) U/L for controls, and mean (+/- SD) AST activity for homozygotes were 502 (+/116) U/L, for heterozygotes, 357 (+/-92) U/L and for controls, 311 (+/-64) U/L and were significantly different between groups (P = 0.04 and P = 0.01 respectively). Resting plasma AST activity was significantly associated with the severity of subsarcolemmal vacuolation (rho = 0.816; P = 0.01) and cytoplasmic inclusions (rho = 0.766; P = 0.01). There were fewer type 2× and more type 2a muscle fibres in PSSM1-affected horses. Our results indicate that PSSM1 has incomplete dominance. Furthermore, the association between plasma muscle enzyme activity and severity of underlying pathology suggests that physical disruption of myofibres may contribute to the myopathic phenotype. This work provides insight into PSSM1 pathogenesis and has implications for related human glycogenoses.
Assuntos
Alelos , Dosagem de Genes , Doenças dos Cavalos/genética , Doenças Musculares/genética , Polissacarídeos/metabolismo , Animais , Biópsia , Doenças dos Cavalos/metabolismo , Doenças dos Cavalos/patologia , Cavalos , Imuno-Histoquímica , Doenças Musculares/metabolismo , Doenças Musculares/patologiaRESUMO
Bovine viral diarrhea virus (BVDV) is one of the most relevant pathogens affecting today's cattle industries. Although great strides have been made in understanding this virus in cattle, little is known about the role of wildlife in the epidemiology of BVDV. While persistently infected cattle are the most important reservoir, free-ranging ungulates may become infected with BVDV as demonstrated by serosurveys and experimental infections. Therefore, free-ranging wildlife may maintain BVDV as the result of an independent cycle and may serve as a reservoir for the virus. Systematic studies on prevalence of BVDV-specific antibodies or frequency of persistent BVDV infection in North American wildlife are sparse, and no information is available from the southeastern United States. The objective of this study was to evaluate blood and skin samples from hunter-harvested white-tailed deer (Odocoileus virginianus) for evidence of BVDV infection. Virus-neutralizing antibodies were detected in 2 of 165 serum samples. Skin biopsy immunohistochemistry (IHC) was performed on samples from 406 deer using a BVDV-specific monoclonal antibody (MAb) (15c5), and BVDV antigen was detected in one sample. A similar IHC staining pattern was obtained using a second BVDV MAb (3.12F1). Viral antigen distribution in the skin sample of this deer resembled that found in persistently infected cattle and in a previously described persistently infected white-tailed deer; thus, the deer was presumed to be persistently infected. Evidence of BVDV infection in free-ranging white-tailed deer should encourage further systematic investigation of the prevalence of BVDV in wildlife.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Cervos/virologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Reservatórios de Doenças/veterinária , Alabama/epidemiologia , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/análise , Doença das Mucosas por Vírus da Diarreia Viral Bovina/sangue , Doença das Mucosas por Vírus da Diarreia Viral Bovina/epidemiologia , Bovinos , Reservatórios de Doenças/virologia , Imuno-Histoquímica/veterinária , Testes de Neutralização/veterinária , Prevalência , Pele/virologiaRESUMO
Bovine viral diarrhea virus (BVDV) affects cattle populations causing clinical signs that range from subclinical immunosuppression to severe reproductive and respiratory problems. Detection and removal of persistently infected (PI) calves is the single most important factor for control and eradication of BVDV. Current testing strategies to detect PI calves rely heavily on immunohistochemistry (IHC) and a commercially available antigen capture ELISA (ACE) assay. These viral assays depend on 1 or 2 monoclonal antibodies which target the E(rns) glycoprotein of BVDV. The sensitivity and specificity of these two tests have been reported previously. The purpose of this research was to characterize a strain of BVDV (AU501) that was undetectable using IHC and ACE based on a single monoclonal antibody, but was consistently detected in samples from a Holstein steer using virus isolation and PCR testing. Sequencing of this AU501 viral isolate revealed a unique mutation in the portion of the genome coding for the E(rns) glycoprotein. This unique field strain of BVDV demonstrates the risk of relying on a single monoclonal antibody for detection of BVDV. Multiple testing strategies, including polyclonal or pooled monoclonal antibodies that detect more than one viral glycoprotein may be necessary to detect all PI calves and facilitate eradication of BVDV.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Glicoproteínas de Membrana/análise , Proteínas do Envelope Viral/análise , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Variação Antigênica , Sequência de Bases , Bovinos , Vírus da Diarreia Viral Bovina/genética , Orelha Externa/virologia , Ensaio de Imunoadsorção Enzimática/veterinária , Imuno-Histoquímica/veterinária , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase/veterinária , Alinhamento de Sequência , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
Bovine viral diarrhea virus (BVDV) infections cause substantial economic losses to the cattle industries. Persistently infected (PI) cattle are the most important reservoir for BVDV. White-tailed deer (Odocoileus virginianus) are the most abundant species of wild ruminants in the United States and contact between cattle and deer is common. If the outcome of fetal infection of white-tailed deer is similar to cattle, PI white-tailed deer may pose a threat to BVDV control programs. The objective of this study was to determine if experimental infection of pregnant white-tailed deer with BVDV would result in the birth of PI offspring. Nine female and one male white-tailed deer were captured and housed at a captive deer isolation facility. After natural mating had occurred, all does were inoculated intranasally at approximately 50 days of pregnancy with 10(6) CCID(50) each of a BVDV 1 (BJ) and BVDV 2 (PA131) strain. Although no clinical signs of BVDV infection were observed or abortions detected, only one pregnancy advanced to term. On day 167 post-inoculation, one doe delivered a live fawn and a mummified fetus. The fawn was translocated to an isolation facility to be hand-raised. The fawn was determined to be PI with BVDV 2 by serial virus isolation from serum and white blood cells, immunohistochemistry on skin biopsy, and RT-PCR. This is the first report of persistent infection of white-tailed deer with BVDV. Further research is needed to assess the impact of PI white-tailed deer on BVDV control programs in cattle.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Cervos/virologia , Vírus da Diarreia Viral Bovina/patogenicidade , Transmissão Vertical de Doenças Infecciosas/veterinária , Complicações Infecciosas na Gravidez/veterinária , Animais , Animais Selvagens/virologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/patologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Bovinos , Cervos/imunologia , Vírus da Diarreia Viral Bovina/imunologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Reservatórios de Doenças/veterinária , Feminino , Sangue Fetal/virologia , Morte Fetal/veterinária , Morte Fetal/virologia , Imuno-Histoquímica/veterinária , Masculino , Testes de Neutralização/veterinária , Gravidez , Complicações Infecciosas na Gravidez/patologia , Complicações Infecciosas na Gravidez/prevenção & controle , Complicações Infecciosas na Gravidez/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Especificidade da EspécieRESUMO
Beef cattle from a herd in north Alabama were examined because of an outbreak of nonfatal skin disease characterized by discrete circumscribed areas of inflammation that developed on the skin from the neck to the hips. Areas of inflammation, which tended to be superficial, underwent necrosis and scabbed over. The scabs eventually dropped off leaving discrete, round, whitish, hairless lesions that were 1.2 to 2.5 cm diameter. Because clinical signs were consistent with those expected with pseudo-lumpy skin disease (PLSD) caused by bovine herpesvirus type 2 (BHV-2), samples from 16 representative animals were submitted for BHV-2 testing. All 16 animals were seropositive for BHV-2, but the virus could not be isolated from skin biopsy specimens or buffy coat samples. Results of a polymerase chain reaction assay incorporating primers designed to amplify 2 DNA sequences from BHV-2 were positive for 3 of the 10 cattle, suggesting that skin lesions in these cattle were a result of PLSD. Our findings suggest that PLSD may be more common and widespread in the United States than suggested by the frequency with which BHV-2 has been isolated from cattle with PLSD-like skin lesions.
Assuntos
Doenças dos Bovinos/diagnóstico , DNA Viral/análise , Herpes Simples/veterinária , Herpesvirus Bovino 2/isolamento & purificação , Reação em Cadeia da Polimerase/veterinária , Alabama/epidemiologia , Animais , Sequência de Bases , Bovinos , Doenças dos Bovinos/epidemiologia , Surtos de Doenças/veterinária , Feminino , Herpes Simples/diagnóstico , Herpes Simples/epidemiologia , Herpesvirus Bovino 2/genética , Doença Nodular Cutânea/diagnóstico , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Pele/patologiaRESUMO
OBJECTIVE: To evaluate persistence of bovine viral diarrhea virus (BVDV) in semen after inoculation of postpubertal bulls. ANIMALS: Three 2-year-old bulls and five 6-month-old calves. PROCEDURE: 3 seronegative 2-year-old bulls were inoculated intranasally with BVDV. Serum and semen samples were obtained at regular intervals until 7 months after inoculation. Serum samples were tested for BVDV by use of virus isolation (VI) and reverse transcription-nested polymerase chain reaction (RT-nPCR) tests. Semen samples were tested for virus by use of VI and RT-nPCR tests. Testicular biopsy specimens were obtained 7 months after inoculation and tested for BVDV by use of immunohistochemical analysis and VI and RT-nPCR tests. Semen samples collected from 1 bull immediately before and 5 and 7 months after inoculation were administered IV to seronegative calves, which were monitored for subsequent viremia and seroconversion. RESULTS: Use of VI and RT-nPCR tests detected transient virus in serum of all bulls. The VI test detected BVDV in semen of 2 bulls for < 21 days after inoculation, whereas RT-nPCR assay detected BVDV until 7 months after inoculation. Virus was detected in testicular biopsy specimens of these 2 bulls by use of immunohistochemical analysis and RT-nPCR assay but could only be isolated from the biopsy specimen of 1 bull. Of the calves administered semen IV to detect infectious virus, only the recipient of semen collected 5 months after inoculation of the adult bull was viremic and seroconverted. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine viral diarrhea virus can persist in semen of acutely infected bulls for several months after exposure.