Tumour cell killing using chemically engineered antibody constructs specific for tumour cells and the complement inhibitor CD59

Immunotherapy using MoAbs is inefficient due to limited activation of human effectors by mouse antibodies and multiple protective mechanisms available to host cells against autologous complement. We have used chemically engineered antibody constructs and human complement in vitro to specifically target and kill neoplastic B lymphoid cells (Raji). Fab′γFcγ² chimaeric antibody (specific for human CD37) was used to activate the classical pathway of human complement on Raji cells, whilst CD59 was neutralized using one of two different bispecific F(ab′γ)² antibody constructs which contained both cell-targeting (anti-CD19 or anti-CD38) and CD59-neutralizing moieties. When either bispecific construct was used to neutralize CD59, 15–25% of cells were lysed. If CD55 was also neutralized using specific antibody, Raji cells were efficiently killed (70% lysis). When added to a mixture of target (Raji) and bystander (K562) cells, one bispecific antibody (anti-CD38 × anti-CD59) could be specifically delivered to Raji, avoiding significant uptake on CD59-expressing bystander cells (K562). The second bispecific antibody (anti-CD19 × anti-CD59) bound equally well to either cell type. Cell-specific targeting was dependent upon combination of a low-affinity anti-CD59 Fab′γ with a high-affinity anti-tumour cell Fab′γ. When Raji and K562 cells were mixed and incubated with a combination of the engineered constructs and anti-CD55 antibodies, Raji cell lysis (30–40%) was observed in the absence of K562 killing. We propose that combinations of these constructs may be of use for treatments such as ex vivo purging of autologous bone marrow or in vivo targeting of tumour cells.     

Linkage relationships between the three phosphoglucomutase loci PGM1, PGM2 and PGM3

1. Phosphoglucomutase phenotypes have been studied in several generations of the family of an individual heterozygous at each of the three loci, PGM₁, PGM₂, and PGM₃. 2. PGM₁ and PGM₂ phenotypes were determined using red cells. Fibroblasts grown in tissue culture were used for PGM₃ phenotyping. 3. The family results support the genetical hypothesis based on the analysis of dizygotic twin pairs that the PGM₃ isozyme patterns found in the placenta are determined by two alleles, PGM¹₃ and PGM²₃. 4. Locus PGM₃ is not closely linked to locus PGM₂ 5. The data also support the previous findings that locus PGM₁ is not closely linked to PGM₂ or PGM₃.     

Striated intramural gallbladder lucencies on US studies: predictors of acute cholecystitis

Ultrasound scans of 51 consecutive patients with gallbladder wall thickening were reviewed, and specific sonographic features were correlated with surgical and clinical follow-up. Two patterns of thickening were identified as specific indicators of the presence or absence of acute cholecystitis. "Striated" wall thickening, consisting of several alternating, irregular, discontinuous, lucent and echogenic bands, was seen in eight of 13 patients (62%) with acute cholecystitis. This pattern was not encountered in any of the patients who did not have acute cholecystitis. Conversely, "three-layer" thickening, consisting of a single circumferential lucent zone between two relatively uniform echogenic layers, was seen in only one of 13 patients (8%) with acute cholecystitis but in 11 of 38 patients (29%) with other diagnoses. Other abnormalities, including the presence of intramural echogenic foci and wall irregularities, were more frequently seen in patients with acute cholecystitis but were not as helpful. Use of these features may suggest or help exclude a diagnosis of acute cholecystitis in those patients in whom the cause of gallbladder wall thickening is otherwise not apparent.     

CONTINUOUS INTERCOSTAL BLOCKADE AFTER CARDIAC SURGERY

The provision of analgesia using continuous bilateral intercostalblockade was compared with that provided by conventional i.v.narcotics for the first 48 h after cardiac surgery. The subjectivequality of analgesia was significantly superior with the regionaltechnique. However, pulmonary function tests, gas exchange,lung volume, and radiological and clinical evidence of pulmonarycomplications were not improved. The failure to reduce morbidityand the potential for complications such as pneumothorax, makesit difficult to recommend the regional analgesia technique inthis situation.     

A technique for eliminating allele specific amplification failure during DNA amplification of heterozygous cells for preimplantation diagnosis

Advances in techniques of molecular biology have made possible the amplification of specific genes from single cells. This has a major clinical application in preimplantation diagnosis of monogenic disorders. However, the incidence of allele specific amplification failure (allele drop out) in heterozygous single cells can lead to misdiagnosis and the transfer of affected embryos. Few studies have been done to investigate the actual cause of allele drop out, although some investigators have succeeded in reducing but not eliminating it. Here we report the efficiency of amplifying both alleles in heterozygous cells lysed according to two different protocols. A total of 177 heterozygous cells from carriers of cystic fibrosis (CF) and haemoglobin C (HbC) were lysed using two different lysis buffers. Interestingly none of the cells that were lysed with sodium dodecyl sulphate/proteinase K showed any example of allele specific amplification failure whereas in those lysed by KOH/dithiothreitol it was present in 17.6 and 4.7% of the CF and HbC cells respectively. Our results suggest that the phenomenon of allele specific amplification failure is at least in part dependent on the lysis buffer used.      

Immunotoxicological Profile of Morphine Sulfate in B6C3F1 Female Mice

This study was undertaken to investigate a number of immuneparameters which may be compromised with exposure to morphinesulfate. Mice were implanted subcutaneously with 8-, 25-, or75-mg morphine sulfate pellets. Placebo pellets of identicalmakeup to the 75-mg morphine pellet (without morphine of course)were used as a control. Twenty-four hours after implantationof a 75-mg morphine pellet, blood levels reached a peak of 1610ng/ml. Corticosterone increased in parallel with morphine andreached a peak level of 966 ng/ml 24 hr after implantation.The dose response of morphine to increase corticosterone, however,was fiat. The weight of the lymphoid organs, spleen and thymus,and the liver were significantly reduced in the morphine-treatedgroups. Morphine treatment was associated with an increase inserum albumin, SGPT, BUN, and alkaline phosphatase indicativeof hepatic damage. In contrast to increased serum proteins,the C3 component of complement was reduced in a dose-dependentmanner. Leukocyte number in the peripheral blood was significantlyreduced, while erythro-cyte number and hematocrit were bothincreased. The number of B cells and T cells was decreased inmorphine-treated animals. However, the percentage of T cellsrelative to B cells was increased. The primary IgM antibodyresponse to the T-depen-dent antigen, sheep red blood cells,was decreased. Natural killer cell activity was reduced in responseto morphine, as was the phagocytic capacity of Kupffer cells.Host-resistance models of Listeria monocytogenes or Streptococcuspneumoniae showed an increased resistance following administrationof morphine. This increased host resistance, however, was notdue to an increase in antimicrobial action of sera obtainedfrom mice treated with morphine. The majority of morphine'seffects on the immune system exhibited a flat dose response,suggesting that these effects may be mediated secondarily throughcorticosterone.     

FIELD TRIAL OF Bacillus thuringiensis var israelensis PELLET FORMULATION IN THE CONTROL OF MOSQUITOES

In a simulated field trial Bacillus thuringiensis var israelensis (BTI) pellet formulation exhibited an enhanced efficacy with increasing dose. A dosage of 1.0 and 1.5 ppm was most effective under simulated field conditions. In field trials persistence of BTI pellet (1.0 ppm) was observed for 35 days in moderately polluted water collection as compared to 21 days in highly polluted water bodies.KEY WORDS: Bacillus thuringiensis, Malaria, Mosquito control     

Gender and Ethnic Differences in Survival in a Cohort of HIV Positive Clients

Objectives: The purpose of this study was to examine gender and ethnic differences in survival of persons receiving treatment for HIV infection to determine if differences existed, and if they did, to assess the possibility of explaining these differences by examining other factors, such as age, disease severity when beginning treatment, alcohol, illicit drugs, tobacco, educational level, living arrangements, antiretroviral treatment, PCP prophylaxis, sexually transmitted diseases, mode of transmission and opportunistic infections.

Design: A retrospective cohort study of all clients receiving treatment at an HIV only clinic from its opening in early 1988 until the end of May 1993. Statistical methods used to examine the data included incidence density ratios, Kaplan‐Meier survival curves, Breslow (generalized Wilcoxon) tests of equality of survival curves and Cox proportional hazards models both with and without time dependent covariates.

Results: In the cohort (37% African American, 7% Hispanic American and 25% female), 220 deaths occurred during 1223 person years of follow‐up. Compared to European American males, the following incidence density ratios were observed: European American females: 0.50, Hispanic American females: 0.70, Hispanic American males: 0.96, African American females: 1.28 and African American males: 2.38. The differences were noted above for gender/ethnicity groups were significant at the p < 0.0001 level. After adjusting for disease stage (as measured by laboratory testing of CD4 positive T‐lymphocytes), educational level, and age, no differences in survival by gender or ethnicity remained. Disease stage and educational level had the greatest prognostic significance.

Conclusions: European Americans entered treatment at a much earlier disease stage (as measured by CD4 positive T‐lymphocyte counts) and had higher educational levels (a surrogate for socioeconomic status) than African Americans. These factors may explain the longer survival in European Americans as compared to African Americans in this cohort.     

A Novel Tertiary Pyridostigmine Derivative [3-N,N-Dimethylcarbamyloxy)-1-Methyl-{Delta}3-Tetrahydropyridine]: Anticholinesterase Properties and Efficacy against Soman

In an effort to develop an effective centrally acting pretreatmentcompound against organophosphorus poisons, the tertiary pyridostigmine(Pyr) derivative 3-(N,N-dimethyI-carbamyloxy)-l-methyl-³-tetrahydropyridine(THP) was synthesized and studied for its anticholinesteraseproperties, as well as its efficacy against soman intoxicationin guinea pigs. Injection of THP (262 µg/kg, im) intoadult male guinea pigs caused inhibition of Wood (30%) and brain(25%) acetylcholinesterase (AChE), showing that THP penetratesthe blood-brain barrier. Pyr (131 µg/kg, im) caused AChEinhibition in the Hood (59%), but not in the brain. The inhibitorypotencies of THP and Pyr were compared by determining theirIC50 values for in vitroinhibition of both AChE (brain, erythrocyte)and pseudo-cholinesterase (plasma) in three mammalian species(guinea pig, rat, rabbit). THP, although effective in inhibitingboth types of cholinesterase, was in general less potent thanPyr. Pretreatment of guinea pigs with THP (262 µg/kg,im) plus Pyr (131 µg/kg, im). 30 min prior to subcutaneoussoman challenge, with no antimuscarink or oxime treatment, protected60% of the animals against 2 ? LD50 of soman. Neither THP norPyr alone was effective. The protective pretreatment regimendid not prevent convulsions, but shortened the recovery timein surviving animals (median recovery time 1.6 hr, comparedto 24 hr in control and other groups of animals pretreated withTHP or Pyr alone). A combination of THP and Pyr thus appearsto provide a means of evaluating the relative importance ofselective peripheral plus central vs peripheral AChE protectionagainst soman.     

Use of Bromoergocryptine in the Validation of Protocols for the Assessment of Mechanisms of Early Pregnancy Loss in the Rat

Use of Bromoergocryptine in the Validation of Protocols forthe Assessment of Mechanisms of Early Pregnancy Loss in theRat. CUMMINGS, A. M., PERREAULT, S. D., AND HARRIS, S. T. (1991).Fundam. Appl. Toxicol 17, 563–574. Validated protocolsfor evaluating maternally mediated mechanisms of early pregnancyfailure in rodents are needed for use in the risk assessmentprocess. To supplement previous efforts in the validation ofa panel of protocols assembled for this purpose, bromoergocryptine(BEC) was used as a model compound because it is known to inhibitpituitary prolactin secretion. BEC was tested using the earlypregnancy protocol (EPP), the decidual cell response technique(DCR), the pre- vs postimplantation protocol (PPP), and embryotransport rate analysis (ETRA). These protocols evaluate theeffects of chemicals on multiple endpoints following exposureduring (a) the first 8 days of pregnancy, (b) early pseudopregnancyaccompanied by decidual induction, (c) the pre- and postimplantationintervals of early pregnancy, or (d) the period of embryo transport.In the EPP, dosing with BEC during Days 1–8 of pregnancyreduced the number of implantation sites found on Day 9 as wellas serum progesterone. The DCR technique revealed a dose-dependentinhibition of decidual growth concomitant with decreased serumprogesterone as a result of BEC treatment. A modified DCR techniqueusing hormone-supplemented ovariectomized rats demonstratedthat BEC did not impair decidual growth in the presence of adequateprogestogenic support. Pie- vs postimplantation dosing indicatedthat implantation is vulnerable to BEC effects at least throughDay 4. BEC had no effect on embryo transport rate. Data fromthese protocols identified BEC as having adverse maternal effectsduring early pregnancy. While the pituitary was not identifiedby these protocols as the site of BECs's primary action, theprotocols did identify a reduction in serum progesterone andimpaired uterine function as toxicological mechanisms mediatingthe reduced fertility seen following BEC exposure.