Programmed death ligand 1 is expressed in canine B cell lymphoma and downregulated by MEK inhibitors.

Programmed death ligand 1 (PD-L1) expression in antigen-presenting cells and tumors can inhibit T cell-mediated immunity. In this study, PD-L1 mRNA and protein expression was evaluated in canine B cell lymphoma (CLL17-71), large T-cell leukemia (CLGL-90), B cell leukemia (GL-1) and primitive leukocyte round cell neoplasia (CLL-1390). Variable PD-L1 mRNA and protein were observed in these cells with high endogenous expression present in CLL17-71 cells. PD-L1 protein was also observed in canine patient B cell lymphoma tissues using immunostaining. PD-L1 and signal transducer and activator of transcription 1 ( STAT1 ) mRNA expression were reduced in the presence of mitogen-activated protein kinase kinase 1.2 (MEK1/2) inhibitors RDEA119 and AZD6244 in CLL 17-71 cells. RDEA119 had similar effect on PD-L1 and STAT-1 in IFN-γ activated CLL-1390 cells. Overall, these results indicate that PD-L1 is expressed in canine B cell lymphoma. Its inhibition by MEK1/2 inhibitors suggests a possible treatment strategy using targeted drugs which likely could enhance antitumor immune response.

A novel eye drop application monitor to assess patient compliance with a prescribed regimen: a pilot study.

PURPOSE: To assess the ability of a novel imaging device to allow physicians to personalize therapeutic regimens based on objective patient drop administration data. METHODS: A novel imaging system was used to record video of the drop technique of subjects in clinic (n=25) or at home (n=17) for 1 week. Video assessment by a reading center was compared with patient reporting and their prescribed regimen with respect to how many drops were applied and how many landed in the eye. RESULTS: Reading center assessment of both drops dispensed and drops landing in the eye was significantly different from the prescribed regimen in the clinic (Pd=0.005, Pi<0.001, respectively) and at-home arms (Pd=0.003, Pi<0.001, respectively). CONCLUSIONS: This imaging system is a powerful tool to help physicians tailor patient therapy more accurately, to help researchers evaluate new drop therapies with objective rather than subjective data, and to potentially facilitate better patient training for improved drug delivery.

Condensation on the posterior surface of silicone intraocular lenses during fluid-air exchange.

PURPOSE: Posterior chamber foldable silicone intraocular lenses (IOLs) are becoming increasingly prevalent in patients undergoing a pars plana vitrectomy with fluid-air exchange. The authors report an important limitation of foldable silicone IOLs during fluid-air exchanges in pars plana vitrectomies. METHODS: The charts of 18 pseudophakic patients with foldable silicone IOLs who underwent vitrectomy with fluid-air exchange by the authors were reviewed. RESULTS: There was a statistically significant difference in the occurrence of condensation during fluid-air exchange between the group of patients with a capsulotomy versus those that did not have a capsulotomy (P = 0.003). Condensation limiting the view of the retina occurred during fluid-air exchange in 11 of 11 of the patients with foldable silicone lenses and a capsulotomy. Attempts to remove the condensation with a soft-tipped aspiration cannula resulted in limited view of the retina for 1 to 2 minutes in 6 of 11 patients. Use of a thin film of silicone oil restored the view in one patient. In the presence of an intact posterior capsule, condensation did not occur on identical foldable silicone IOLs in seven of seven patients. CONCLUSION: Recognition of the presence of a foldable silicone lens is important when an air-fluid exchange is anticipated. If a capsulotomy is present, the surgeon must be aware that condensation may limit the view of the retina severely during and after surgery. Intraoperatively, the view of the retina usually can be restored in short surgeries by wiping the posterior lens surface with a soft-tipped cannula, and in more complex surgeries by applying a thin film of silicone oil on the posterior surface of the lens.

An anti-gp41 human monoclonal antibody that enhances HIV-1 infection in the absence of complement.

B lymphocytes from tonsillar tissue of an asymptomatic HIV-1-seropositive subject were transformed with Epstein-Barr virus (EBV) and tested for the production of HIV-1-specific antibodies by ELISA, using purified HIV-1SF2.2F11, a monoclonal antibody derived from a transformed line, is of the IgG1 subclass and recognizes an epitope in the conserved region of the envelope transmembrane glycoprotein gp41, which is expressed on the surface of HIV-infected T cells. The antibody does not mediate the lysis of infected T cells in antibody-dependent cellular cytotoxicity (ADCC) assays and does not neutralize the infectivity of HIV-1SF2 or the homologous isolate HIV-1TT2.2F11 appears to be the first anti-gp41 human monoclonal antibody that enhances the infectivity of an HIV-1 strain (i.e., SF128A) in the absence of complement.

Postmortem stability of dopamine D1 receptor mRNA and D1 receptors.

The effects of postmortem interval on dopamine D1 mRNA and D1 receptors were assessed in rat striatum under conditions simulating the handling of human brain tissue at 0, 6, 12, and 24 h postmortem. The amount of D1 mRNA was measured by both in situ hybridization film and emulsion autoradiography with [35S]dATP-labeled oligonucleotide probes. D1 receptor density was determined by autoradiography with [125I]SCH 23982. Neither the total amount of D1 mRNA in the striatum nor the frequency distribution of striatal cells expressing D1 mRNA varied with the postmortem interval. There was a modest but significant decrease (ca. 10%) in D1 receptors over the 24 h postmortem interval; this decrease occurred within the first 6 h postmortem, with no further decreases up to 24 h postmortem. These findings suggest that the effects of postmortem interval on D1 mRNA and receptors are minimal and should not limit an examination of possible alterations in dopamine D1 receptor mRNA and D1 receptors in the postmortem brains of humans with neuropsychiatric disease.

In vitro cytotoxic targeting by human mononuclear cells and bispecific antibody 2B1, recognizing c-erbB-2 protooncogene product and Fc gamma receptor III.

Bispecific murine monoclonal antibody 2B1, possessing dual specificity for the human c-erbB-2 protooncogene product and human Fc gamma receptor III (CD16) was evaluated for the ability to promote specific lysis of c-erbB-2-positive tumor cells in vitro. In short-term 51Cr release assays with human mononuclear cells as effectors and SK-Br-3 human breast cancer cells as targets, neither parental antibody of 2B1 mediated significant specific lysis, but bispecific antibody was as active as a chemical heteroconjugate, with 5 ng/ml of 2B1 causing half-maximal lysis at an effector/target ratio of 20:1 and 2 ng/ml 2B1 causing half-maximal lysis at an E/T ratio of 40:1. The cytotoxic targeting activity of 2B1 F(ab')2 fragment was the same as that of whole bispecific antibody, and the activity of whole 2B1 was not reduced when assays were performed in 100% autologous human serum, indicating that 2B1 binds effector cells through the CD16-binding site derived from parental antibody 3G8 rather than through its Fc portion. Variable inhibition of 2B1-mediated lysis was observed when autologous polymorphonuclear leukocytes from different donors were added to mononuclear effector cells at a 2:1 ratio; this inhibition was overcome at higher antibody concentration. 2B1 bispecific monoclonal antibody was also able to mediate targeted cytolysis using whole human blood as a source of effector cells or using effector or target cells derived from ovarian cancer patients.

Selection of hybrid hybridomas by flow cytometry using a new combination of fluorescent vital stains.

A new combination of fluorescent dyes (rhodamine 123 and hydroethidine) was used to internally label hybridoma fusion partners. Murine hybridoma 520C9 (recognizing human c-erbB-2) was labeled with hydroethidine. Murine hybridoma 3G8 (recognizing human Fc gamma receptor III) was labeled with rhodamine 123, and verapamil was used to block rhodamine efflux via P-glycoprotein. Viability assays showed little cytotoxicity from these dyes at the concentrations used. The labeled cells were fused with polyethylene glycol, sorted for dual fluorescence on an Epics V cell sorter, and cloned. Hybrid hybridomas producing bispecific antibodies were selected for ability to promote lysis of SK-Br-3 breast cancer cells by human mononuclear cells. Several positive clones were obtained and shown to have a double content of DNA. Bispecific antibody produced by subclone 2B1 was purified by anion exchange chromatography and shown to bind both tumor cells and Fc gamma R III bearing cells. Using two parameter flow cytometric analysis, we were able to measure a 'bridging' effect of this bispecific antibody, which caused formation of complexes between PMNs and SK-Br-3 cells. Either parental antibody could compete with bispecific antibody to block such complexing. This fusion method provides several advantages over other techniques presently used (speed, convenience, low toxicity and automatic exclusion of dead cells) and can be applied to produce other hybrid hybridomas.

Synthesis and in vitro evaluation of 2,3-dimethoxy-5-(fluoroalkyl)-substituted benzamides: high-affinity ligands for CNS dopamine D2 receptors.

A number of 2,3-dimethoxy-5-(fluoroalkyl)-N-[(1-ethyl-2- pyrrolidinyl)methyl]benzamides (with or without a 6-hydroxy group) were synthesized and evaluated as dopamine D2 receptor ligands. The parent acids were synthesized via the Claisen rearrangement of the appropriate O-allyl ethers, which were derived from o-vanillic acid or 2,3-dimethoxysalicylic acid. A decrease in reactivity was found to be characteristic of pentasubstituted benzoates, and difficulties were encountered with the introduction of fluorine onto the ethyl side chains. The (fluoroethyl)- and (fluoropropyl)salicylamides were 5 times more potent than the corresponding benzamides in inhibiting [3H]spiperone binding to the D2 receptor. These (fluoroalkyl)salicylamides are of potential value for in vivo positron emission tomography (PET) studies upon the basis of their relatively selective, high potency binding affinity for the D2 receptor.

Distribution and physical properties of BCA200, a Mr 200,000 glycoprotein selectively associated with human breast cancer.

Of 122 mouse monoclonal antibodies selective for human breast cancer, 13 immunoprecipitated an acidic glycoprotein from SK-Br-3 and ZR-75-30 human breast cancer cells. The antigen (BCA200) migrates with an apparent molecular weight of 200,000 on reducing and 180,000 on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a single polypeptide chain with a folded domain stabilized by a disulfide bond. Cross-blocking and sandwich immunoassays detected at least three distinct antigenic determinants on BCA200. Scatchard experiments measured 1,000,000 to 5,000,000 antigen copies per SK-Br-3 cell. The tissue distribution of BCA200 was studied using two monoclonals to different epitopes. Neither antibody stained any cells in human blood. When frozen sections of 20 normal human tissues were immunoperoxidase stained, the only positive structures were mucinous glands of colon, transitional epithelium of bladder, sweat glands of skin, and acinar epithelium of breast. Antibody 454C11 stained 16 of 21 breast tumor frozen sections and 9 of 12 breast cancer cell lines, while antibody 520C9 stained 5 of 20 breast tumors and 4 of 10 breast cancer lines. Cross-reaction was observed with lung, prostatic, pancreatic, endometrial, and ovarian cancer, but not with lymphoma, melanoma, colon, stomach, bladder, or esophageal cancer. When conjugated to ricin A chain, 10 of 13 antibodies produced immunotoxins selectively cytotoxic to SK-Br-3 breast cancer cells.

Measurement of retinal blood vessel width using computerized image analysis.

We report a new method for quantitating retinal blood vessel width from fluorescein angiogram negatives using a computerized image analyser. Interuser and intrauser variability were 3 to 4 times lower than reported with older methods. In addition, model blood vessels were used to test the accuracy of our method under four clinically relevant conditions: variations in vessel width, fluorescein concentration, flash intensity and background fluorescence. Although computed measurements were affected by vessel width, fluorescein concentration and flash intensity, the linearity of the actual versus computed width was maintained during wide variations in all four conditions (r = 0.95, P less than 0.0001). Accuracy was best achieved at a flash intensity of 150 W/sec and a fluorescein concentration of 45 micrograms/ml. The results of this study provide a better understanding of factors affecting the apparent width of retinal blood vessels in fluorescein angiograms. This technique should be useful in rapidly obtaining accurate measurements of blood vessel width from fluorescein angiograms.

Pulmonary hypertension secondary to serum hyperviscosity in a patient with rheumatoid arthritis.

A patient with rheumatoid arthritis who was evaluated for dyspnea of six months' duration is described. Although no primary cardiac or parenchymal lung disease was identified, right heart catheterization revealed marked pulmonary hypertension. The patient was presumed to have pulmonary arteritis. Evaluation of her hyperproteinemia, however, led to the discovery of a polyclonal gammopathy with a marked increase in plasma viscosity. Although the classic clinical findings of the hyperviscosity syndrome were minimal, the patient underwent plasmapheresis, resulting in a marked reduction of pulmonary artery pressures (from 53 +/- 4 mm Hg, mean +/- SD, to 30 +/- 3 mm Hg, p less than 0.05) and pulmonary vascular resistance (from 707 +/- 63 dynes/second/cm5 to 421 +/- 72 dynes/second/cm5, p less than 0.05) concomitant with a return to normal plasma viscosity. Her dyspnea completely resolved. This represents the first successful treatment of pulmonary hypertension by plasmapheresis. Protein evaluation revealed the presence of intermediate complexes of IgG rheumatoid factor. The hyperviscosity syndrome should be considered in the differential diagnosis of pulmonary hypertension in patients with rheumatoid arthritis and other disorders associated with a polyclonal or monoclonal gammopathy. Pulmonary hypertension secondary to the hyperviscosity syndrome is reversible by plasmapheresis. Immunosuppressive therapy that reduces immunoglobulin production may provide a means of long-term treatment.