Expression of “early” and “late” viral functions in a somatic cell hybrid between a mouse cell and a spontaneous yielder SV 40-transformed Chinese hamster cell

Summary A somatic cell hybrid (Cl. 6d) was originated from the fusion of mouse (3T3-4E) and spontaneous yielder SV 40-transformed Chinese hamster (CHK/SVLP AG) cells. During the early stages of its history, the Cl. 6d hybrid underwent a rapid chromosome loss, preferentially loosing hamster chromosomes. This was not a constant tendency of the hybrid cells.As the parental CHK/SVLP AG cells, the hybrid cells were always found 100 per cent SV 40 T-antigen positive. While in CHK/SVLP AG cells infectious SV 40 DNA, V-antigen and virus were regularly detected, in the hybrid cells only infectious DNA was occasionally detected. This was not due either to the loss of an essential Chinese hamster gene(s) or to the presence of an inhibiting mouse cell component(s); it was apparently the consequence of inability of the cells to properly activate the resident SV 40 genome(s).After superinfection with SV 40 DNA, the hybrid cells—though capable of synthesizing SV 40 V-antigen—were unable to ensure virus assembly. Experimental evidence was obtained suggesting that SV 40 maturation is dependent on a cellular function(s).With 1 Figure     

Coxsackievirus B3-associated myocardial pathology and viral load reduced by recombinant soluble human decay-accelerating factor in mice

Coxsackievirus B3 (CVB3) infection can result in myocarditis, which in turn may lead to a protracted immune response and subsequent dilated cardiomyopathy. Human decay-accelerating factor (DAF), a binding receptor for CVB3, was synthesized as a soluble IgG1-Fc fusion protein (DAF-Fc). In vitro, DAF-Fc was able to inhibit complement activity and block infection by CVB3, although blockade of infection varied widely among strains of CVB3. To determine the effects of DAF-Fc in vivo, 40 adolescent A/J mice were infected with a myopathic strain of CVB3 and given DAF-Fc treatment 3 days before infection, during infection, or 3 days after infection; the mice were compared with virus alone and sham-infected animals. Sections of heart, spleen, kidney, pancreas, and liver were stained with hematoxylin and eosin and submitted to in situ hybridization for both positive-strand and negative-strand viral RNA to determine the extent of myocarditis and viral infection, respectively. Salient histopathologic features, including myocardial lesion area, cell death, calcification and inflammatory cell infiltration, pancreatitis, and hepatitis were scored without knowledge of the experimental groups. DAF-Fc treatment of mice either preceding or concurrent with CVB3 infection resulted in a significant decrease in myocardial lesion area and cell death and a reduction in the presence of viral RNA. All DAF-Fc treatment groups had reduced infectious CVB3 recoverable from the heart after infection. DAF-Fc may be a novel therapeutic agent for active myocarditis and acute dilated cardiomyopathy if given early in the infectious period, although more studies are needed to determine its mechanism and efficacy.     

Nucleolar organizer regions in transitional cell tumors of the bladder

The traditional silver staining technique that identifies nucleolar organizer region-associated proteins has recently been adapted and applied to paraffin sections, and used to examine changes in nucleolar organizer region numbers in a variety of benign and malignant conditions. We describe herein the enumeration of nucleolar organizer regions in a variety of benign and malignant conditions of the transitional epithelium of the urinary bladder. While there is a statistical separation between normal and neoplastic groups, there is a degree of overlap between normal, inflammatory, dysplastic, and neoplastic urothelium.     

A genetic study of hyper-alpha-lipoproteinemia

Because of its association with longevity and reduced incidence of coronary heart disease, it becomes important to find out how elevated HDL-cholesterol levels are determined. Analyses of family data from Cincinnati initially suggested environmental factors common to sibs; however, some form of dominant inheritance could not be ruled out. Reanalysis of the Cincinnati data by Iselius and Lalouel concluded against a major locus, but did identify three families as possibly segregating for a major locus. Analysis of an additional 26 kindreds from the same population in Cincinnati by Siervogel and associates concluded that a major gene could be causing familial aggregation of high density lipoprotein in white kindreds. In this analysis, we pooled all the white Cincinnati kindreds (n = 31), and investigated the familial transmission using complex segregation analysis. We failed to obtain clear evidence for major locus determination. Under the parsimonious hypothesis of no major locus, the polygenic heritability and common sibling environmental correlation were estimated as 0.531 and 0.263, respectively, consistent with other evidence.     

Babesia bovis merozoite surface protein-2c (MSA-2c) contains highly immunogenic,conserved B-cell epitopes that elicit neutralization-sensitive antibodies in cattle

The search for vaccine candidates against bovine babesiosis caused by Babesia bovis is greatly focused on the identification of merozoite surface-exposed antigens that are widely conserved, functionally relevant and immunodominant in cattle protected against B. bovis infections. We have recently identified msa-2c, a member of the B. bovis variable merozoite surface antigen (VMSA) gene family, which in contrast to other members, appears to be highly conserved among geographically distant B. bovis strains. In this study, we further investigated the potential of the msa-2c gene product as diagnostic and vaccine candidate for bovine babesiosis. RT-PCR studies demonstrated that MSA-2c is transcribed in merozoites of the Argentine R1A strain. In addition, antibodies against R1A recombinant MSA-2c reacted in immunoblots with a single protein of approximately 30kDa in B. bovis merozoite extracts from both R1A and Australian "S" strains, demonstrating translation of this protein in these two strains and conservation of B-cell epitopes between them. These antibodies reacted with the cell surface of R1A merozoites in fixed immunofluorescence assays, indicating the surface localization of MSA-2c. This localization was confirmed by live immunofluorescence studies in two different strains, R1A and S2P. These results also demonstrate the conservation of MSA-2c surface-exposed B-cell epitopes between these two strains. Sera from cattle either naturally or experimentally infected with Argentine strains of B. bovis specifically recognized rMSA-2c in immunoblots, reinforcing the idea that B-cell epitopes in rMSA-2c are widely conserved among field strains of B. bovis. Furthermore, our results show that these B-cell epitopes are highly immunogenic, suggesting that MSA-2c may be a useful diagnostic tool for the detection of bovine babesiosis by B. bovis. Experimental vaccination of five bovines with rMSA-2c resulted in elicitation of high specific anti-rMSA-2c IgG titers, with similar amounts of IgG(1) and IgG(2) produced. Importantly, bovine anti-rMSA-2c antibodies were able to neutralize in vitro bovine erythrocyte invasion by R1A merozoites suggesting a significant functional role for MSA-2c. Taken together these results postulate MSA-2c as a candidate for the development of novel tools for improved control of bovine babesiosis.     

Cellular distribution of bovine leukemia virus proteins gp51SU, Pr72(env), and Pr66(gag-pro) in persistently infected cells

Monoclonal antibodies (mAbs) against bovine leukemia virus (BLV) mature proteins and precursors were used to map the localization of these proteins in persistently infected non-lymphocytic cell lines using immunofluorescence assay (IFA) and immuno-electron microscopy. IFA staining was observed in the basolateral surface of live FLK-BLV cells. When using a mAb against Pr66(gag-pro), mottled pinpoint fluorescence was seen in the cell surface of polarized cells, but no reaction was observed in cells undergoing mitosis. However, a mAb against Pr72(env) stained only mitotic cells and cellular fragments. Additionally, in these dividing cells, this envelope (Env) precursor polyprotein was not evenly distributed but concentrated predominantly in only one daughter cell. To the best of our knowledge, this observation has not been reported previously, either for BLV or for other retroviruses. The results of immunogold electron microscopy confirmed the specificity of the mAbs in the intracellular level. In infected cells, Pr72(env) and gp51SU were seen in proximity at the plasma membrane in incipient budding sites. Additionally, the mAb against Pr72(env) also reacted with Env precursor polyproteins in the mitochondria of BLV-bat(2) ultrathin sections. These mAbs may be used as a tool for mapping virus excretion sites in the cell surface of naturally or in vitro infected cells in the different stages of the cell cycle.     

Fine-needle aspiration diagnosis of Kaposi's sarcoma in a developing country

Fine-needle aspiration (FNA) cytology was performed on 15 patients with peripheral lymphadenopathy and/or skin lesions referred to the Department of Pathology of the Hospital Central of Maputo, Maputo, Mozambique. Epitrochlear lymph nodes were the most frequently aspirated site. All aspirates allowed diagnoses of Kaposi's sarcoma (KS). Smears contained loosely cohesive clusters of bland spindle cells, with a radial arrangement and nuclear crush artifacts. These diagnostic clues have not been described in other spindle-cell intranodal lesions that should be considered in differential diagnoses. Taking into consideration the high prevalence of AIDS and limited resources for diagnosis in Africa, FNA cytology appears to be a useful method for the diagnosis of KS in developing countries, reducing the necessity for surgical lymph node excision.     

Efficacy and Safety of Atezolizumab Plus Bevacizumab Following Disease Progression on Atezolizumab or Sunitinib Monotherapy in Patients with Metastatic Renal Cell Carcinoma in IMmotion150: A Randomized Phase 2 Clinical Trial

BackgroundThe use of immune checkpoint inhibitors combined with vascular endothelial growth factor (VEGF)-targeted therapy as second-line treatment for metastatic clear cell renal cancer (mRCC) has not been evaluated prospectively.ObjectiveTo evaluate the efficacy and safety of atezolizumab + bevacizumab following disease progression on atezolizumab or sunitinib monotherapy in patients with mRCC.Design, setting, and participantsIMmotion150 was a multicenter, randomized, open-label, phase 2 study of patients with untreated mRCC. Patients randomized to the atezolizumab or sunitinib arm who had investigator-assessed progression as per RECIST 1.1 could be treated with second-line atezolizumab + bevacizumab.InterventionPatients received atezolizumab 1200 mg intravenously (IV) plus bevacizumab 15 mg/kg IV every 3 wk following disease progression on either atezolizumab or sunitinib monotherapy.Outcome measurements and statistical analysisThe secondary endpoints analyzed during the second-line part of IMmotion150 included objective response rate (ORR), progression-free survival (PFS), and safety. PFS was examined using Kaplan-Meier methods.Results and limitationsFifty-nine patients in the atezolizumab arm and 78 in the sunitinib arm were eligible, and 103 initiated second-line atezolizumab + bevacizumab (atezolizumab arm, n = 44; sunitinib arm, n = 59). ORR (95% confidence interval [CI]) was 27% (19–37%). The median PFS (95% CI) from the start of second line was 8.7 (5.6–13.7) mo. The median event follow-up duration was 19.4 (12.9–21.9) mo among the 25 patients without a PFS event. Eighty-six (83%) patients had treatment-related adverse events; 31 of 103 (30%) had grade 3/4 events. Limitations were the small sample size and selection for progressors.ConclusionsThe atezolizumab + bevacizumab combination had activity and was tolerable in patients with progression on atezolizumab or sunitinib. Further studies are needed to investigate sequencing strategies in mRCC.Patient summaryPatients with advanced kidney cancer whose disease had worsened during treatment with atezolizumab or sunitinib began second-line treatment with atezolizumab + bevacizumab. Tumors shrank in more than one-quarter of patients treated with this combination, and side effects were manageable.