In vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells stimulated with synthetic peptides

A protocol for in vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells (PBMCs) was developed. Antigen presenting cells (APCs) consisted of Staphylococcus aureus Cowan-I (SAC-I) activated PBMCs treated with a citrate-phosphate buffer at pH 3 to release endogenous peptides bound to surface MHC. This treatment resulted in transient expression of empty class I molecules which could be subsequently stabilized with peptide and beta 2-microglobulin (beta 2m). SAC-I activated PBMCs from HLA-A2.1 normal donors loaded with HBV core 18-27 peptide following acid treatment were used to stimulate PBMCs depleted of CD4+ T cells, in the presence of recombinant interleukin-7 (rIL-7). After 12 days, cells were restimulated with autologous, peptide-pulsed, adherent cells and tested for CTL activity 7 days later. In 23 independent experiments from 13 different HLA-A2.1 donors, this protocol resulted in induction of primary CTL more than 90% of the time. As indicated by both the frequency and magnitude of the response against peptide-sensitized target cells, SAC-I activated PBMCs treated with acid were the most efficient stimulator APC. Thirteen per cent of the cultures generated were capable of lysing target cells transfected with the HBV core antigen and, in general, these CTL cultures exhibited high avidity for the HBV core peptide. This protocol is generally applicable to different antigens and class I alleles, and thus, may be utilized to screen large numbers of peptides to identify human CTL epitopes.     

Diagnosis of pleural effusions. Experience with clinical studies, 1986 to 1990

OBJECTIVES: To identify in patients with pleural effusion which procedures are most useful in separating malignant from nonmalignant pleural effusions and to identify which procedures most commonly lead to a definitive diagnosis. DESIGN: Prospective consecutive case series. SETTING: Pulmonary referral hospital in Prague, Czech Republic. PATIENTS: One hundred seventy-one adults between ages 18 and 70 years with a pleural effusion and a Karnofsky score of 70 or above. INTERVENTIONS: All patients underwent history, physical, pleural fluid cytologic study, laboratory evaluation of serum and pleural fluid, pleural biopsy, bronchoscopy, and lung scan and/or pulmonary arteriogram. RESULTS: In this series in which 45% of the patients had malignant effusions, 19% had paramalignant effusions, and 36% had benign diseases, the pleural fluid cytologic study was the best for establishing a diagnosis. The pleural fluid carcinoembryonic antigen (CEA) levels above 10 had a high specificity (90%) for malignancy but had low sensitivity (37%). The pleural fluid CEA level was increased only in 19% of patients with paramalignant effusions. Although there were statistically significant differences in the mean results on several biochemical tests of pleural fluid, none were very accurate in separating malignant from benign disease. CONCLUSION: From this study, we conclude that patients with an undiagnosed pleural effusion should be evaluated in an individualized stepwise manner. If malignancy is strongly considered, the initial three steps should be relatively noninvasive and include clinical evaluation and cytologic study.     

Recombinant bovine somatotropin increases milk yield and calf gain in diverse breeds of beef cattle: associated changes in hormones and indices of metabolism

In Exp. 1, Angus (A, n = 30), Charolais (C, n = 37), and Simmental (S, n = 30) multiparous cows received (s.c.) recombinantly derived bovine somatotropin (bST; sometribove, 500 mg) or vehicle (VEH) at 2-wk intervals from 124 to 228 d postpartum (DPP). Calves were weaned at 228 DPP. Bovine somatotropin increased (P < .01) milk yield and percentage of milk fat similarly in A, C, and S cows. Calf weaning weight was greater (P < .05) in cows treated with bST than in those given VEH. Administration of bST decreased deposition of fat and increased concentrations of IGF-I, insulin, glucose, and nonesterified fatty acids. In Exp. 2, we compared effects of bST initiated before or after the breeding season. Charolais (n = 33) and S (n = 40) cows were administered (at 2-wk intervals) VEH or bST beginning at 28 DPP (B-bST) or bST beginning at 105 DPP (A-bST). Calves were weaned at 243 DPP. Administration of bST before or after the breeding season increased milk yield on DPP 136 and 194; however, yields were greater in A-bST than in B-bST cows. Milk yields were similar in all cows at 236 DPP, corresponding to decreased forage availability. Calf body weight was greater (P < .05) in A-bST than VEH; B-bST calves were similar to VEH. Fat depth was greater in VEH than in bST-treated cows in C but not in S cows. Serum IGF-I was greater in A- and B-bST than in VEH cows. Mean days from calving to serum progesterone > 1 ng/mL and pregnancy rates were similar in VEH, A-, and B-bST cows. Administration of bST increased cow milk yield and subsequent calf weaning weight when initiated after 100 d postpartum. As anticipated, bST increased IGF-I, insulin, glucose, and nonesterified fatty acids. Administration of bST before and during the breeding season did not affect reproductive performance.     

Analysis of the nucleotide sequence of the treehopper-transmitted geminivirus, tomato pseudo-curly top virus, suggests a recombinant origin

The genome of tomato pseudo-curly top virus (TPCTV), originating from Florida, has been cloned and sequenced. TPCTV is the only geminivirus identified with a vector specificity which falls outside the Cicadellidae (leafhoppers) and Aleyrodidae (whiteflies). Infectivity of the cloned viral genome was demonstrated by Agrobacterium-mediated inoculation of several host species. Progeny virus was transmissible by the treehopper vector of TPCTV, Micrutalis malleifera (Fowler). The genome of TPCTV shows features typical of both subgroups I and III genera of the family Geminiviridae. The coat protein of TPCTV, although distinct from all previously characterized geminiviruses, exhibits features more akin to the leafhopper-transmitted geminiviruses than those transmissible by the whitefly Bemisia tabaci Genn. The relationship of TPCTV to other geminiviruses, particularly beet curly top virus, is discussed in relation to the possible evolutionary origins of this virus.     

FDA points-to-consider documents: the need for dietary control for the reduction of experimental variability within animal assays and the use of dietary restriction to achieve dietary control

Wild type p53 protein has been shown by recent investigations to be involved in the negative regulation of cell proliferation, whereas aberrant p53 protein has lost this negative regulation of cell growth. Wild type p53 protein, which has a very short half-life, has generally been considered to be undetectable using immunohistochemical methods; however, according to a recent report, wild type p53 protein may accumulate in the nuclei because of a defective ubiquitin pathway. Aberrant p53 protein has a longer half-life, and thus is visible using immunohistochemical methods. In this study, both the proliferative potential represented by the MIB-1 staining index (SI) and the immunoreactivity of p53 protein in 51 intracranial meningiomas were studied applying immunohistochemical staining methods to archival paraffin sections. The correlation among MIB-1 SI, p53 immunoreactivity, histopathologic findings and the clinical course of the meningiomas was also analyzed retrospectively. Although it is not possible with available reagents to distinguish between aberrant p53 protein and wild type p53 protein, statistical analyses show that p53 protein was immunostained both in meningiomas with high MIB-1 SI and in recurrent meningiomas. This demonstrates the close relationship among p53 immunoreactivity, MIB-1 SI, and recurrence; therefore, the presence of p53 protein by immunohistochemical examination may suggest the proliferative activity of meningioma and is capable of serving as a predictor of future recurrence.