Disabled infectious single-cycle herpes simplex virus as an oncolytic vector for immunotherapy of colorectal cancer.

New modalities of treatment for colorectal cancer are required to support and improve those currently available. One such approach is immunotherapy by transfer of immunostimulatory genes to tumor cells. Here, we report the use of a herpes simplex virus (HSV) vector that is capable of a single round of infection (disabled infectious single-cycle [DISC]-HSV) as a gene transfer vehicle for colorectal cancer. This vector has potential advantages over other vectors for cancer immunotherapy in that it lyses infected tumor cells. Infection with DISC-HSV inhibited tumor cell growth both in vitro and in vivo. In addition, DISC-HSV-mediated cell killing occurs by both apoptotic and necrotic mechanisms. A range of colorectal tumor cell lines could be rapidly transduced with DISC-HSV/lacZ (14-90% in 4 hr). Both tumor prevention and tumor therapy protocols showed clear antitumor effects with DISC-HSV/mGM-CSF. In the prophylactic approach, an infected/irradiated whole cell vaccine protected up to 80% of mice from rechallenge. In addition, intratumoral injection of established tumors with DISC-HSV/GM-CSF caused rejection in 40% of mice and generated some protection from subsequent rechallenge. In both cases, however, it is clear that a dominant therapeutic effect of the DISC-HSV vector derives from its oncolytic properties, irrespective of the transduced gene. As a prelude to taking these studies forward to human clinical trials, we demonstrate that tumor cells could be successfully grown from freshly obtained human colorectal cancer resections (within 1 week of surgery), were transduced with DISC-HSV/hGM-CSF, and secreted the cytokine. This study provides the preclinical basis for trials of immunotherapy of colorectal cancer using DISC-HSV.

Routine antenatal Rhesus D immunoglobulin prophylaxis: the results of a prospective 10 year study.

OBJECTIVE: To assess the clinical and financial impact, and identify the problems, of providing routine antenatal RhD immunoglobulin prophylaxis for Rhesus D negative nulliparae. DESIGN: A retrospective (1980-1986) and prospective (1987-1996) comparison between two similar populations, one population with nulliparae offered routine RhD immunoglobulin 500 IU prophylaxis at 28 and 34 weeks of gestation part way through the study period, and the other population not offered prophylaxis at any time. SETTING: Obstetric units in two counties (three health districts) with similar annual numbers of maternities and the Regional Blood Transfusion Service antenatal serology laboratory. PARTICIPANTS: Non-sensitised Rhesus D negative pregnant nulliparae. INTERVENTIONS: Intramuscular RhD immunoglobulin 500 IU at 28 and 34 weeks of gestation to eligible women booked for confinement in one county; the intervention not offered in the other county. MAIN OUTCOME MEASURES: 1. Rhesus D sensitised second pregnancy rate; 2. success in providing prophylaxis to eligible women; 3. serology laboratory activity changes; 4. potential savings from the prophylaxis programme. RESULTS: Prophylaxis significantly reduced iso-immunisation in the next pregnancy when compared with historical (OR 0.28, CI 0.14-0.53; P < 0.0001) and contemporary controls (OR 0.43, CI 0.22-0.86; P = 0.02). However, success at achieving comprehensive prophylaxis was disappointing, with only 89% of eligible women receiving the first injection, 74% both injections, and for only 29% were both at the correct gestation. Fifty-two percent of women delivered after 40 weeks of gestation, beyond the period of adequate prophylaxis protection. The savings in antenatal interventions, neonatal care and possible long term ill-health that result from very preterm birth should be considerable. CONCLUSION: Routine prophylaxis for nulliparae significantly reduces the incidence of sensitised next pregnancies with consequent savings, and its adoption nationwide should be encouraged. A programme offering antenatal prophylaxis for all Rhesus D negative women is unlikely to be economic. Improvement in uptake of prophylaxis is needed; alternative administration strategies should be explored.

A novel herpes vector for the high-efficiency transduction of normal and malignant human hematopoietic cells.

Herpes simplex viruses (HSVs) would offer numerous advantages as vectors for gene transfer, but as yet they have not proved capable of transducing hematopoietic cells. Using a genetically inactivated form of HSV that is restricted to a single cycle of replication (disabled single-cycle virus, [DISC-HSV]), we have transduced normal human hematopoietic progenitor cells and primary leukemia blasts with efficiencies ranging from 80% to 100%, in the absence of growth factors or stromal support. Toxicity was low, with 70% to 100% of cells surviving the transduction process. Peak expression of transferred genes occurred at 24 to 48 hours after transduction with the DISC-HSV vector, declining to near background levels by 14 days. Despite this limitation, sufficient protein is produced by the inserted gene to permit consideration of the vector for applications in which transient expression is adequate. One example is the transfer of immunostimulatory genes, to generate leukemia immunogens. Thus, murine A20 leukemia cells transduced with a DISC-HSV vector encoding granulocyte-macrophage colony-stimulating factor were able to stimulate a potent antitumor response in mice, even against pre-existing leukemia. The exceptional transducing ability of the DISC-HSV vector should therefore facilitate genetic manipulation of normal and malignant human hematopoietic cells for biological and clinical investigation.

Effect of issuing an invitation for breast cancer screening to women aged 65 to 69.

OBJECTIVE: Breast cancer screening is available to all women over 50 in the United Kingdom on the National Health Service breast screening programme. Only women aged between 50 and 64 receive a written invitation, with women over 64 being expected to self refer. Unfortunately, despite the higher incidence of breast cancer in the older age group only a very small proportion of women over 64 attend for screening. The aim of the study was to test the hypothesis that women between the ages of 65 and 69 will attend for screening if invited. METHOD: Women aged 65 to 69 were invited for screening from general practices in the Inverness area and were treated in all respects as the usual screening age group. RESULTS: An uptake of 76% was achieved with a cancer detection rate of 9.3 per thousand. CONCLUSION: These results lend weight to the suggestion that women in this age group should receive a specific invitation for breast cancer screening.

The role of serum amylase in the diagnosis of acute pelvic inflammatory disease.

The use of serum amylase levels in the diagnosis of acute pelvic inflammatory disease (PID) was investigated prospectively. Eighty-five women presenting with the chief complaint of lower abdominal pain were entered into the study; all patients were examined by one of the principal investigators. In addition to the usual laboratory studies, a serum amylase level was obtained on all patients; the investigators were blinded to the results. Patients were diagnosed with PID if they fulfilled previously published clinical criteria. Forty-eight patients met the criteria for the diagnosis of PID (PID group); 37 patients were diagnosed with other disease processes (non-PID group). The average serum amylase level for the PID group was 62 U/L, with a standard deviation (STD) of 24; for the non-PID group, the average was 76 U/L with an STD of 32. Although there was a statistical difference between the two groups (P < .05), there was no clinically significant difference because both values fell within the normal range of serum amylase. The routine use of serum amylase in the diagnosis of acute PID seems to be of no value.

Neonatal alloimmune thrombocytopenia due to anti-HPA-5b (Br(a), Zav(a), Hca): the importance of third-generation platelet antibody detection techniques, a case report.

Investigation of the maternal serum in a case of suspected alloimmune neonatal thrombocytopenia by conventional, second-generation platelet serological assays (platelet radioactive antiglobulin test [PRAT], platelet suspension immunofluorescence test [PSIFT] and solid-phase adherence assay (SPAA, 'Capture-P') demonstrated only the presence of HLA class-I antibodies of limited specificities: no platelet-specific antibodies were detectable. The use of a third generation, glycoprotein capture assay (monoclonal antibody-specific immobilization of platelet antigens, MAIPA) revealed the additional presence of anti-HPA-5b with a titre of 1 in 32. Despite this relatively high titre, and the fact that it was able to induce a prolonged thrombocytopenia, this antibody was not detectable by conventional assays. In view of these findings we conclude that the use of MAIPA is essential when investigating cases of suspected alloimmune neonatal thrombocytopenia.

Post transfusion septicaemia 1980-1989: importance of donor arm cleansing.

AIMS: To determine the prevalence of Pseudomonas fluorescens on the arms of blood donors, and to elucidate one possible cause for its predominance (60% of cases during 1980-89) in exogenous post transfusion septicaemia (PTS). METHODS: Skin swabs were taken from the arms of 782 blood donors and cultured on to heated blood agar. After incubation, Oxidase reagent and the Gram stain were used to select non-fermentative Gram negative rods, which were then subcultured and identified using the Analytical Profile System (API) 20 NE system. RESULTS: Non-fermentative Gram negative rods were found on the arms of 11.7% of donors, Pseudomonas spp on 1.0%, and Ps fluorescens on the arms of 0.3% of donors. CONCLUSIONS: This evidence emphasises the absolute requirement for efficient skin cleansing of blood donors' arms to minimise the risk of exogenous PTS.

Serological screening tests for syphilis in pregnancy: results of a five year study (1983-87) in the Oxford region.

Between 1983 and 1987, 62 out of 76519 pregnancies in 51 mothers had a positive miniaturised Treponema pallidum haemagglutination assay (TPHA) test--1 in 1234, or 0.81 per 1000 births. About two thirds of these mothers had syphilis and the remainder non-venereal treponematoses such as yaws or pinta. Antenatal screening identified 13 patients with previously unknown acquired syphilis, 11 of whom were given antibiotics during pregnancy. There were six fetal losses among the 62 TPHA positive pregnancies, but none had evidence of congenital syphilis. No live born child in this study group showed stigmata of congenital syphilis. It is concluded that despite the current low incidence of syphilis in the United Kingdom it is imperative to continue antenatal serological screening and to emphasise the importance of early adequate treatment of the infection.

In-utero intravascular transfusion of the fetus for the management of severe Rhesus isoimmunization--a reappraisal.

Ten fetuses, severely affected by Rhesus (D) haemolytic disease, received one to three intravascular blood transfusions at between 18 and 30 weeks gestation, with the use of fetoscopically guided needles into one of the umbilical cord vessels. Although the technique was successfully accomplished in all cases, the fetal response to the procedure was varied. Only two fetuses survived beyond the neonatal period, and one child subsequently died principally because of the problems resulting from premature delivery. The reason for the low rate of survival has been explored and the continued use of the method described is now questioned.

Pre-transfusion non-invasive quality assessment of stored platelet concentrates.

The Platelet Monitor System has been designed to assess routinely prepared platelet concentrates (P.C.) during storage. The system is based upon observations of changes in light transmission induced by platelets in intact packs. It is practical, provides constant gentle agitation for P.C., and non-invasively give continuous semi-quantitative evidence of the probable numbers of discoid platelets within each pack. The information yielded correlates both with established in vitro parameters of platelet behaviour, and with in vivo survival of platelets in normal healthy volunteers. The device safely provides the Blood Banker with the means to achieve on-going assessment of methods and improvements--or failings--in P.C. production. To the clinician it offers information throughout and even beyond present arbitrary storage limits, about which P.C. are likely to be of least or greatest value to a thrombocytopenic patient.

Blood group antibody screening tests during pregnancy.

In a 2-year period 667 sera from approximately 70,000 (0.95%) antenatal patients were found to contain 726 atypical red blood cell antibodies. Overall, 66% of the immunized mothers were rhesus (D) positive. Apart from four antibody specificities to rhesus system antigens, knowledge of the rhesus (D) group gave no guide to the ability of the patients to form any of the remaining 21 specificities encountered. Of the 726 antibodies 221 (30%) were not detected in the initial sample tested and 50 of the 92 patients who produced antibodies during pregnancy had not developed detectable antibody when they were sampled at 28 weeks. The significance of these late onset antibodies is discussed both in relation to the risk of haemolytic disease in the newborn and transfusion reactions in the mother. An optimum protocol for testing is defined which takes account of antibody production during the pregnancy and use of this protocol constitutes an attempt to combine maximum clinical safety with minimal consumption of resources.

Separation of plasma from whole blood by membrane filtration in oscillatory flows.

This paper describes the performance of a microfiltration plasmapheresis unit operating with reversing oscillatory flows. The device consists of a flat channel duct between cellulose nitrate membranes and was used to extract plasma from anticoagulated fresh whole bovine blood. Measurements were made of plasma flux, haematocrit concentration, haemolysis and protein sieving coefficients. The effects on plasma flux are reported for alterations in the stroke and frequency of flow pulsations, transmembrane pressure, membrane properties and blood throughput. It was found that the imposition of oscillatory flows enhanced the plasma extraction rate by a factor of 3, producing about 0.9 litre/min/m2 membrane.

Comparative trial of six methods for the detection of CMV antibody in blood donors.

Six techniques were compared to find the most suitable method for determining the cytomegalovirus (CMV) antibody status of blood donors. Five hundred and ninety-six random sera were tested by immunofluorescence (IF), complement fixation (CFT), two enzyme linked immunosorbent assays (ELISA), a commercial indirect haemagglutination test (IHA)--used as supplied, and a locally devised micromodification of the same IHA test. Five hundred and thirty-five sera shared total agreement of results by all tests. The ELISA tests were the most discordant with other methods (10.5% discordancies both positive and negative). IF and CFT correlated well with other tests (0.8% discordances each) but for different reasons are unsatisfactory for donor screening. The IHA test used as supplied and its micromodification gave the most consistent results (0.8% and 0.5% discordancies respectively). The micromodification is easy to perform and read; it compares very favourably with CFT and IF for material costs and expertise required, and readily lends itself to the testing of large numbers of sera in a reasonable time. Within certain provisos the micro-IHA technique described is recommended as the most suitable test for blood donor screening.