Discovery of natural-product-derived sequanamycins as potent oral anti-tuberculosis agents.

The emergence of drug-resistant tuberculosis has created an urgent need for new anti-tubercular agents. Here, we report the discovery of a series of macrolides called sequanamycins with outstanding in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb). Sequanamycins are bacterial ribosome inhibitors that interact with the ribosome in a similar manner to classic macrolides like erythromycin and clarithromycin, but with binding characteristics that allow them to overcome the inherent macrolide resistance of Mtb. Structures of the ribosome with bound inhibitors were used to optimize sequanamycin to produce the advanced lead compound SEQ-9. SEQ-9 was efficacious in mouse models of acute and chronic TB as a single agent, and it demonstrated bactericidal activity in a murine TB infection model in combination with other TB drugs. These results support further investigation of this series as TB clinical candidates, with the potential for use in new regimens against drug-susceptible and drug-resistant TB.

[Cardiac amyloidosis: pathological classification and clinical analysis of 48 cases].

Objective: To investigate the histological features and clinical manifestations in different types of cardiac amyloidosis to improve diagnostic accuracy. Methods: The histopathological features and clinical manifestations of 48 patients diagnosed with cardiac amyloidosis by Congo red stain and electron microscopy through endomyocardial biopsy were collected in West China Hospital of Sichuan University from January 2018 to December 2021. Immunohistochemical stains for immunoglobulin light chains (κ and λ) and transthyretin protein were carried out, and a review of literature was made. Results: The patients age ranged from 42 to 79 years (mean 56 years) and the male to female ratio was 1.1 to 1.0. The positive rate of endomyocardial biopsy was 97.9% (47/48), which was significantly higher than that of the abdominal wall fat (7/17). Congo red staining and electron microscopy were positive in 97.9% (47/48) and 93.5% (43/46), respectively. Immunohistochemical stains showed 32 cases (68.1%) were light chain type (AL-CA), including 31 cases of AL-λ type and 1 case of AL-κ type; 9 cases (19.1%) were transthyretin protein type (ATTR-CA); and 6 cases (12.8%) were not classified. There was no significant difference in the deposition pattern of amyloid between different types (P>0.05). Clinical data showed that ATTR-CA patients had less involvement of 2 or more organs and lower N-terminal pro-B-type natriuretic peptide (NT-proBNP) than the other type patients (P<0.05). The left ventricular stroke volume and right ventricular ejection fraction of ATTR-CA patients were better than the other patients (P<0.05). Follow-up data of 45 patients was obtained, and the overall mean survival time was 15.6±2.0 months. Univariate survival analysis showed that ATTR-CA patients had a better prognosis, while cardiac amyloidosis patients with higher cardiac function grade, NT-proBNP >6 000 ng/L, and troponin T >70 ng/L had a worse prognosis (P<0.05). Multivariate survival analysis showed that NT-proBNP and cardiac function grade were independent prognostic factors for cardiac amyloidosis patients. Conclusions: AL-λ is the most common type of cardiac amyloidosis in this group. Congo red staining combined with electron microscopy can significantly improve the diagnosis of cardiac amyloidosis. The clinical manifestations and prognosis of each type are different and can be classified based on immunostaining profile. However, there are still a few cases that cannot be typed; hence mass spectrometry is recommended if feasible.

[Bladder cancer local staging about muscle invasion: 3.0T MRI performance following transurethral resection].

OBJECTIVE: To evaluate the performance of 3.0T magnetic resonance imaging examination (MRI) for the local detecting of muscle invasive bladder cancer following transurethral resection of bladder tumor (TURBT). METHODS: Retrospective study identified 55 patients with pathology-proven bladder cancer who underwent transurethral resection of bladder tumor followed by 3.0T magnetic resonance imaging between September 2012 and April 2019 in our hospital. Two radiologists reviewed pelvic magnetic resonance imaging together and judged muscle invasive bladder cancer. Sensitivity, specificity and accuracy were calculated for the presence of muscle invasion by T2 weighted imaging (T2WI) only, diffusion-weighted imaging (DWI) only and T2WI+DWI compared with the findings at radical cystectomy as the reference standard. RESULTS: Of the 55 patients with pathological results from radical cystectomy, 3.64% (2/55) had no residual disease; 29.09% (16/55) were non-muscle invasive bladder cancer on pathology, including 13 cases in T1 and 3 cases in Ta; 34.55% (19/55) were in stage T2 depending on pathology, 25.45% (14/55) in T3, and 7.27% (4/55) in T4. The average age was 60.76 years, ranging from 42 to 82 years. There were 48 males and 7 females in our study. Before pelvic MRI examination, all the patients received transurethral resection of bladder tumor, including 16 cases taking the operation in our hospital and 39 cases in other hospitals. The interval between the pelvic MRI examination and transurethral resection of bladder tumor was more than 2 weeks in all the patients. They all underwent radical cystectomy within 1 month after the pelvic MRI examination, and no patient underwent radiotherapy or chemotherapy in our study during the interval between the MRI examination and radical cystectomy. T2WI only, DWI only, and T2WI+DWI of 3.0T magnetic resonance imaging for readers were with sensitivity: 94.59%, 83.78%, 91.89%; with specificity: 66.67%, 77.78%, 72.22% and with accuracy: 85.45%, 81.82%, 85.45%, respectively. CONCLUSION: 3.0T MRI may have a role in diagnosing muscle invasive bladder cancer following TURBT. T2WI has the advantage of detecting the location of bladder tumor, and DWI has the advantage of differentiating between the benign and malignant lesion. 3.0T MRI T2WI+DWI has a good utility in the detection of muscle invasive bladder cancer following TURBT with satisfied accuracy.

[Effects of patient-controlled intravenous analgesia with butorphanol versus sufentanil on early postoperative rehabilitation following radical laparoscopic nephrectomy].

Objective: To investigate the effects of patient-controlled intravenous analgesia with butorphanol versus sufentanil on early postoperative rehabilitation following radical laparoscopic nephrectomy. Methods: One hundred patients undergoing radical laparoscopic nephrectomy in Affiliated Cancer Hospital of Zhengzhou University from September 2018 to February 2020 were divided into two groups (n=50) using a random number table: butorphanol patient-controlled intravenous analgesia group (group A) and sufentanil patient-controlled intravenous analgesia group (group B). Patient-controlled intravenous analgesia (PCIA) was performed at the end of surgery. The formulation of group A was butorphanol (0.15 mg/kg) and ketorolac tromethamine (180 mg) using the physiological saline at a dilution of 100 ml. The formulation of group B was sufentanil (1.5 µg/kg) and ketorolac tromethamine (180 mg) using the physiological saline at a dilution of 100 ml. At the time points of 4, 8, 24, 48 h after operation (T(1), T(2), T(3), T(4)), VAS scores at rest and cough were recorded. The incidence of remedial analgesia, the number of pressings during 48 h after the operation, the postoperative anal exhaust recovery time of the patients were recorded. Quality of recovery-40(QoR-40) scores were recorded at T(3) and T(4). Adverse reactions were recorded. Results: There was no significant difference in VAS scores at rest and cough at T(1), T(2), T(3) and T(4) between two groups (all P>0.05). There was no significant difference in the incidence of remedial analgesia and the number of pressings during 48 h after the operation between two groups (all P>0.05). The postoperative anal exhaust recovery time of the patients in group A was (32±6) h, which was lower than that in group B with statistically significant difference [(40±5) h, t=7.937, P<0.01]. The QoR-40 total scores in group A were higher than those in group B at T(3) and T(4), which were (185.8±2.5) vs (170.7±2.7), (194.8±1.9) vs (183.6±2.6), and the differences were statistically significant (t=28.878, 25.025, all P<0.01). The incidence of nausea, retching/vomiting, respiratory depression and itch during 48 h after the operation in group A were 10%, 6%, 2%, 2%, which were lower than that in group B (32%, 20%, 14%, 18%), with statistically significant difference (χ(2)=7.294, 4.322, 4.891, 5.983, all P<0.05). Conclusion: PCIA with butorphanol or sufentanil can provide satisfactory analgesia for patients undergoing radical laparoscopic nephrectomy, but butorphanol can promote postoperative rehabilitation with fewer adverse reactions.

[The correlation between left atrial sphericity and thromboembolic events in patients with atrial fibrillation].

Objective: To study the correlation between left atrial sphericity (LASP) and thromboembolic events (TE) in patients with atrial fibrillation (AF). Methods: This study was conducted in patients with AF underwent radiofrequency ablation in the Department of Cardiology of First Affiliated Hospital of Zhengzhou University from January 2011 to October 2018. The AF patients with TE (study group, n=157) and the AF patients without TE (control group, n=157) were matched for age and gender. The differences of LASP and other related indexes between the two groups were compared, and the correlation between LASP and TE was analyzed by conditional logistic regression. The receiver operating characteristic (ROC) curve was drawn to analyze the diagnostic value of LASP for TE. Results: (1) The LASP in the study group was significantly higher than that in the control group [ (87.5±7.1) % vs. (82.8±6.1) %, P=0.001]. (2) Conditional logistic regression analyses showed that LASP (OR=1.10, 95%CI 1.05-1.16, P=0.001), left atrial volume index (OR=1.01, 95%CI 1.00-1.02, P=0.016) and CHA(2)D-VASc score (OR=1.77, 95%CI 1.30-2.41, P=0.001) were independently and positively correlated with TE. (3) The ROC curve analysis showed that the area under the curve (AUC) of left atrial sphericity (AUC=0.712, 95%CI 0.656-0.768, P=0.001) was larger than the AUC of either left atrial volume index (AUC=0.650, 95%CI 0.589-0.710, P=0.001) or CHA(2)D-VASc score (AUC=0.612, 95%CI 0.550-0.674, P=0.001). (4) CHA(2)D-VASc-LASP(2) score was positively correlated with TE (OR=1.95, 95%CI 1.55-2.42, P=0.001). Conclusion: LASP is independently and positively correlated with TE in patients with AF.

[Retrospectively analysis on 95 postpartum hemorrhage women treated with obstetrics and gynecology in intensive care unit].

Objective: To review the clinical aspects and pathogenesis of postpartum hemorrhage (PPH) and investigate the optimal protocols for intervention. Methods: From February 2009 to December 2015, data of normal labour and casearean birth women admitted to intensive care unit (ICU) in our hospital because of hematobilia were selected. 95 patients were divided into three groups (e. g ≥500-1 000 ml, ≥1 000-1 500 ml, ≥1 500-2 500 ml and ≥2 500 ml group) according to the bleeding volume. A retrospective analysis was performed to study the pathogenesis of PPH, organ function, surgical intervention and clinical prognosis on hemorrhage. Results: The data comprised 20 504 women over the 6-year period. 95 (0.463%) of which resulted in PPH and were admitted to ICU. 9 of these patients with PPH unsurvived. The value of creatinine and urea nitrogen, the score of APACHE Ⅱ and the possibility of multiple organ dysfunction syndromethe (MODS) increased with the amount of bleeding (P<0.05). For patients with PPH caused by injury of birth canal and/or placenta factors, there was significant difference among three groups on amount of bleeding (P<0.05). For patients with surgical intervention such as vaginal packing, interventional treatment and exploratory laparotomy conducted in 6 hours, the volume of transfusion was(759±114) ml. The volume of transfusion was (2 000±829) ml and (4 999±1 699) ml in 6 to 12 hours intervention group and in greater than 12 hours intervention group, respectively. The volume of transfusion significant increased over intervention time. There was a statistically significant difference in all groups (P<0.05). Conclusions: Classified treatment should be conducted according the classification on the amount of bleeding. Patients with severe PPH and/or tendency of organ failure should be admitted to ICU. Measures for maintenance of the function of organs are necessary, while appropriate surgical intervention is also needed based on the cooperation between ICU and obstetrical department, and the cure rate could be improved.

[Analysis on the nuclear division index of peripheral blood lymphocytes in the 281 general population of Anhui, China].

OBJECTIVE: To investigate the reference range and influeing factors of the nuclear division index (NDI) of peripheral blood lymphocyte in Chinese general population in Anhui province. METHODS: We selected 281 subjects from the general poulation in Anhui province, without occupational exposure to genetic toxicants and no chronic disease history. We used questionnaires to collect occupational history, age, gender, region, body mass index, smoking, and alcohol drinking status etc. NDI was measured by cytokinesis block micronucleus assay in peripheral blood lymphocytes, and the related factors were also analyzed. And NDI was used as the dependent variable, age, gender and other factors as independent variables to conduct stepwise multiple linear regression. RESULTS: We found the data of NDI according with normal distribution, and the nuclear division index was 1.71±0.22, the minimum value was 1.10 while the maximum was 2.36. The results showed that NDI value of the males (1.67±0.20) were lower than that of the females (1.76±0.24), the difference was statistically significant (t=-3.65, P<0.001); current smokers NDI (1.66±0.18) lower than non-smokers (1.73±0.24) differences were statistically significant (t=3.06, P=0.002); the NDI of drinking groups (1.66±0.20) was lower than that of non-drinking population (1.74±0.23), the differences was statistically significant (t=3.15, P=0.002); Using multiple stepwise linear regression calibration factors and found that gender was an independent factor of NDI (ß=0.098, Sx=0.027, t=3.66, P< 0.001). CONCLUSION: We set the reference value on the nuclear division index among general population of survey areas in this study, it could provide a reference for similar studies and will provide reference for better evaluation of the effects of hazards on the body.

In vitro and in vivo activities of the nitroimidazole TBA-354 against Mycobacterium tuberculosis.

Nitroimidazoles are a promising new class of antitubercular agents. The nitroimidazo-oxazole delamanid (OPC-67683, Deltyba) is in phase III trials for the treatment of multidrug-resistant tuberculosis, while the nitroimidazo-oxazine PA-824 is entering phase III for drug-sensitive and drug-resistant tuberculosis. TBA-354 (SN31354[(S)-2-nitro-6-((6-(4-trifluoromethoxy)phenyl)pyridine-3-yl)methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine]) is a pyridine-containing biaryl compound with exceptional efficacy against chronic murine tuberculosis and favorable bioavailability in preliminary rodent studies. It was selected as a potential next-generation antituberculosis nitroimidazole following an extensive medicinal chemistry effort. Here, we further evaluate the pharmacokinetic properties and activity of TBA-354 against Mycobacterium tuberculosis. TBA-354 is narrow spectrum and bactericidal in vitro against replicating and nonreplicating Mycobacterium tuberculosis, with potency similar to that of delamanid and greater than that of PA-824. The addition of serum protein or albumin does not significantly alter this activity. TBA-354 maintains activity against Mycobacterium tuberculosis H37Rv isogenic monoresistant strains and clinical drug-sensitive and drug-resistant isolates. Spontaneous resistant mutants appear at a frequency of 3 × 10(-7). In vitro studies and in vivo studies in mice confirm that TBA-354 has high bioavailability and a long elimination half-life. In vitro studies suggest a low risk of drug-drug interactions. Low-dose aerosol infection models of acute and chronic murine tuberculosis reveal time- and dose-dependent in vivo bactericidal activity that is at least as potent as that of delamanid and more potent than that of PA-824. Its superior potency and pharmacokinetic profile that predicts suitability for once-daily oral dosing suggest that TBA-354 be studied further for its potential as a next-generation nitroimidazole.

Analysis of signaling pathways in recurrent breast cancer.

Breast cancer remains the second largest cause of death in women from cancer. By analyzing gene expression profiles in samples from breast cancer patients, 844 differentially expressed genes (DEGs) were identified in breast cancer metastasis. The 10 most significant signaling pathways identified through enrichment analysis contained DEGs were involved in oxidative phosphorylation, DNA replication, extracellular matrix-receptor interactions and others. Furthermore, survival analysis demonstrated that 5 of these signaling pathways were closely related to the survival time of breast cancer patients including basal transcription factors, cell cycle, ECM-receptor interaction, spliceosome, and DNA replication. Our findings increase the understanding of the network of signaling pathways involved in breast cancer metastasis and may provide theoretical support for further therapeutic study.

A novel monoclonal antibody against human CD80 and its immune protection in a mouse lupus-like disease.

Blockade of the interactions between CD28/CTLA-4 and their ligands, CD80 (B7, B7.1)/CD86 (B70, B7.2), is an attractive means to induce antigen-specific peripheral tolerance in autoimmune disease and organ transplantation. In this study, we generated and characterized a monoclonal antibody (Clone 4E5) against human CD80. 4E5 could recognize both human and mouse CD80 and suppress mixed lymphocyte reaction in vitro. To investigate their potency for clinical use, we further administrated 4E5 to a mouse lupus-like disease model (C57BL/J6) induced by Pristane. 4E5 could inhibit the immune response and attenuate the severity of lupus-like disease. The data showed 4E5 function and suggested that blockade of CD80/CD28 co-stimulatory signal pathway with 4E5 is a promising strategy to decelerate the progression of lupus-like disease and other autoimmune diseases.

Synthesis and structure-activity relationships of 2-aryl-4-oxazolylmethoxy benzylglycines and 2-aryl-4-thiazolylmethoxy benzylglycines as novel, potent PPARalpha selective activators- PPARalpha and PPARgamma selectivity modulation.

The synthesis and follow-up SAR studies of our development candidate 1 by incorporating 2-aryl-4-oxazolylmethoxy and 2-aryl-4-thiazolylmethoxy moieties into the oxybenzylglycine framework of the PPARalpha/gamma dual agonist muraglitazar is described. SAR studies indicate that different substituents on the aryloxazole/thiazole moieties as well as the choice of carbamate substituent on the glycine moiety can significantly modulate the selectivity of PPARalpha versus PPARgamma. Potent, highly selective PPARalpha activators 2a and 2l, as well as PPARalpha activators with significant PPARgamma activity, such as 2s, were identified. The in vivo pharmacology of these compounds in preclinical animal models as well as their ADME profiles are discussed.

Pressure-dependent electronic structures in multiferroic DyMnO(3): A combined lifetime-broadening-suppressed x-ray absorption spectroscopy and ab initio electronic structure study.

Variations in the electronic structure and structural distortion in multiferroic DyMnO(3) were probed by synchrotron x-ray diffraction, lifetime-broadening-suppressed x-ray absorption spectroscopy (XAS), and ab initio electronic structure calculations. The refined x-ray diffraction data enabled an observation of a diminished local Jahn-Teller distortion of Mn sites within MnO(6) octahedra in DyMnO(3) on applying the hydrostatic pressure. The intensity of the white line in Mn K-edge x-ray absorption spectra of DyMnO(3) progressively increased with the increasing pressure. With the increasing hydrostatic pressure, the absorption threshold of an Mn K-edge spectra of DyMnO(3) shifted toward a greater energy, whereas the pre-edge line slightly shifted to a smaller energy. We provide the spectral evidence for the pressure-induced bandwidth broadening for manganites. The intensity enhancement of the white line in Mn K-edge spectra is attributed to a diminished Jahn-Teller distortion of MnO(6) octahedra in compressed DyMnO(3). A comparison of the pressure-dependent XAS spectra with the ab initio electronic structure calculations and full calculations of multiple scattering using the code FDMNES shows the satisfactory agreement between experimental and calculated Mn K-edge spectra.

LncRNA UCA1 regulates cervical cancer survival and EMT occurrence by targeting miR-155.

OBJECTIVE: Cervical cancer rate is increasing recently. LncRNA UCA1 plays a role in gynecological tumors, but its expression and mechanism in cervical cancer have not yet been elucidated. PATIENTS AND METHODS: The tumor tissues and adjacent tissues of cervical cancer patients were collected to measure LncRNA UCA1 and miR-155 level by Real-time PCR. The Luciferase report analyzed the relationship between LncRNA UCA1 and miR-155. HeLa cells were separated into NC group, UCA1 siRNA group, UCA1 siRNA + miR-155 inhibitor group followed by analysis of cell proliferation, invasion and migration and EMT-related genes E-cadherin and Vimentin expression by Real time PCR. RESULTS: UCA1 level was elevated and miR-155 was reduced in cervical cancer tissues with significant differences compared to adjacent tissues (p <0.05). UCA1 was negatively correlated with miR-155 level (p <0.05). Patients with high UCA1 level showed short survival time (p <0.05). Down-regulation of UCA1 can significantly inhibit cell proliferation, migration and invasion. It can also increase E-cadherin expression and decrease Vimentin expression (p <0.05). MiR-155 is a target miRNA of UCA1. MiR-155 inhibitor can significantly reverse UCA1 siRNA's effect (p <0.05). CONCLUSIONS: UCA1 expression in cervical cancer is increased and related to patient survival and miR-155 expression is reduced. Lnc-RNA UCA1 regulates EMT occurrence in cervical cancer cells by targeting miR-155.

Temperature dependent pinning phenomenon in superconducting Nb films with triangular and honeycomb pinning arrays.

The pinning phenomena in superconducting Nb films with triangular and honeycomb pinning arrays were explored. Special temperature dependent phenomena were found for both films. For the film with a triangular pinning array, the pronounced matching peaks in the critical currents as a function of magnetic field reduce from six to three within a narrow temperature range. This temperature dependent matching effect is explained by considering the dramatic change of coherent length with temperature when the temperature is close to T(c). In order to compare with the film with a triangular pinning array, we fabricated a film with a honeycomb pinning array with similar pinning site spacing and pinning size. Special prominent matching peaks at H = 3.5H(1) were found for this film. Molecular dynamic simulations were made to study this phenomenon. The ground state distribution of vortices obtained from simulations reasonably explains the prominent matching peaks. Pronounced temperature dependent matching effects were also found for the film with a honeycomb pinning array.

[Clinical significance of HLA-A, -B, -C, -DRB1, -DQB1 haplotype gene frequencies].

Objective: To analyze family-based haplotype frequencies of HLA-A, -B, -C, -DRB1 and -DQB1 genes and their clinical significance. Methods: The data of HLA genotyping in 3568 families undergoing related haploidentical transplantation between 2012 and 2017 at the First Affiliated Hospital of Soochow University were retrospectively evaluated. The HLA genotyping was performed by PCR amplification with sequence-based typing (PCR-SBT) and sequence-specific oligonucleotide probe (PCR-SSOP) methods. The family genetic analysis and haplotype frequencies were also investigated. Results: All the families were divided into 3 groups, including group1 of 1 422 entire families; group2 of 1 310 patients and either of their parents or one of their children; group3 of 836 patients and their HLA≥5/10 matched sibling donors. In the haplotypes with frequencies greater than 0.1% in group1+ group2, the frequency of A*11∶01-B*40∶01-C*03∶04-DRB1*11∶01-DQB1*03∶01, A*02∶07-B*51∶01-C*14∶02-DRB1*09:01-DQB1*03∶03 were significantly different between group1 and group2 (P=0.029, 0.033) . The frequency of A*11∶01-B*46∶01-C*01∶02∶01G-DRB1*09∶01-DQB1*03∶03 was significantly different between group1 and group3 (P=0.035) . The frequency of A*02∶01-B*40∶01-C*07∶02-DRB1*09∶01-DQB1*03∶03 was significantly different between group1 and group2 (P=0.034) , or group1 and group3 (P=0.034) . The frequency of A*24∶02-B*13∶01-C*03∶04-DRB1*12∶02-DQB1*03:01 was significantly different between group2 and group3 (P=0.046) . Conclusion: In this study, we summarize the prevalence of haplotype frequencies in terms of HLA-A, -B, -C, -DRB1 and-DQB1. Based on the database of family haplotype analysis, patients and donor candidates are sorted with matched HLA genotype while unmatched HLA haplotype. Even in patients without entire family information, HLA haplotype analysis assists in choosing the optimal related or unrelated donors.

Inhibitory effects of phytohemagglutinin on growth of leukemia lymphoblasts L5178Y in vitro and in vivo.

Phytohemagglutinin (PHA) inhibited the growth of mouse leukemia lymphoblasts L5178Y in culture. RNA synthesis was repressed significantly within the first hour, and the rates of DNA and protein synthesis were significantly decreased within the second hour; maximum inhibition occurred after 24 hours. The ability of the cells to form colonies in soft agar was limited to a greater extent at much lower PHA concentrations. The cells lost their tumorigenicity in syngeneic DBA/2 mice after 4 hours of PHA treatment, when the viability was not appreciably decreased. The cultures of a PHA-resistant strain derived from a resistant colony cloned from the PHA-containing soft agar plate showed increased resistance toward PHA. A fourfold increase in PHA concentration was needed for the same rate of inhibition of the original cell line to be attained.

Identification and physicochemical characterization of a tumor-associated antigen from canine transmissible venereal sarcoma.

A tumor antigen associated wtih canine transmissible venereal sarcoma (CTVS) has been identified and partially characterized. The antigen was demonstrated in 3-M KCI and saline extracts of washed CTVS cells. Rabbit anti-CTVS antisera absorbed wih glutaraldehyde cross-linked normal dog serum and pooled homogenates of canine spleen, thymus, lymph node, and liver were used to identify the antigen. The specificity of the rabbit anti-CTVS antiserum for the CTVS antigen was established by use of immunodiffusion, two-dimensional electrophoresis, and the enzyme-linked immunosorbent assay (ELISA). Extracted CTVS antigen was fractionated by Sephadex G-200 chromatography, and the antigen factions were identified with the use of absorbed rabbit anti-CTVS antiserum and the ELISA technique. Antigen activity was detected in samples with estimated molecular weights greater than 70,000 daltons. Antigen fractions reacted with absorbed anit-CTVS antiserum to form a single precipitation band in immunodiffusion studies. Extraction of CTVS antigen with 3 M KCl and saline in the presence of a protease inhibitor did not significantly alter the antigen activity or molecular weight of the CVTS antigen. Cytoplasmic and nucleolar fluorescence were observed in an indirect immunofluorescence test with the use of acetone-fixed CTVS cells and absorbed anti-CTVS antiserum. The CTVs antigen activity was greatly reduced by trypsin digestion, by incubation for 1 hour at 65 degrees C, and by exposure to pH 2.8, 4.0, and 11.0 for 1 hour each.

Immunoelectron microscopic localization of a tumor-associated antigen (TAA) in the canine transmissible venereal sarcoma by anti-TAA-antiferritin hybrid antibodies.

Immunoferritin and immunoperoxidase reagents were used to localize a tumor-associated antigen (TAA) of the canine transmissible venereal sarcoma (CTVS). Round tumor cells from the CTVS at different stages of growth, i.e., progressive, steady state, and regressing, had TAA diffusely distributed throughout the cytoplasm. In general, TAA was not found on the plasma membrane, within the nucleus, between inner and outer membranes, in cytoplasmic vacuoles, or specifically with any part of the cytocavitary system. Transitional tumor cells, which are intermediate cell types between round cells and spindle-shaped cells and which appear in the tumors at steady state and regressing stages, contained less TAA in their cytoplasm than did the round cell type. The microvilli of tumor cells also contained TAA, suggesting that, in addition to whole cell lysis, shedding of all or parts of these processes may be a mechanism of TAA release as evidenced by the presence of antigenic activity in the extracellular material.

Canine transmissible venereal sarcoma: quantitation of T-lymphocyte subpopulations during progressive growth and spontaneous tumor regression.

The levels of T-lymphocyte subpopulations expressing IgMFc and IgGFc receptors (i.e., T mu lymphocytes and T gamma lymphocytes, respectively) were quantitated for T-lymphocytes obtained from peripheral blood, draining and nondraining lymph nodes, and the tumor mass during progressive growth and spontaneous regression of the canine transmissible venereal sarcoma. Analysis of the T-lymphocyte subpopulations in these lymphoid compartments demonstrated that distinct profiles of T mu and T gamma lymphocytes correlated respectively with the growth and regression statuses of the tumor. The percent of T gamma lymphocytes in the peripheral blood of dogs with progressing, steady-state, and late-regressing tumors was significantly increased over that of control dogs (P less than .05, P less than .001, and P less than .001, respectively) and over that of dogs with early-regressing tumors (P less than .05, P less than .01, and P less than .02, respectively). The percent of T gamma lymphocytes in the lymph nodes draining the tumor was observed to have a significant increase in dogs with progressing (P less than .01), steady-state (P less than .01), and late-regressing (P less than .001) tumors compared with that in control dogs. The percentage of T gamma lymphocytes was observed to have a significant increase in the nondraining lymph nodes of dogs with steady-state and late-regressing tumors compared with that of control dogs (P less than .01 and P less than .002, respectively) and that of dogs with progressing tumors (P less than .001 and P less than .0005, respectively). The percent of tumor-infiltrating T gamma lymphocytes was lowest in tumors that were growing progressively. A significant increase in T gamma lymphocytes was observed in steady-state (P less than .05), early-regressing (P less than .001), and late-regressing (P less than .05) tumors. Early-regressing tumors contained significantly (P less than .005) greater levels of T gamma lymphocytes than did late-regressing tumors.

Stimulatory effect of 3', 5'-adenosine monophosphate on protein synthesis in heat-shocked murine leukemia lymphoblasts.

The murine leukemia lymphoblast line L5178Y was tested for its early molecular response to the presence of dibutyryl cyclic AMP. Protein synthesis was reversibly inhibited by short durations of hyperthermia, and against this reduced background of protein synthetic activity, early leucine incorporation (30-120 min) was markedly and consistently stimulated by the presence of the cyclic nucleotide. The effect became one of inhibition with extended times of incubation (longer than 120 min).