Human cytomegalovirus (HCMV) in clinical material cannot replicate efficiently in vitro until it has adapted by mutation. Consequently, wild-type HCMV differ fundamentally from the passaged strains used for research. To generate a genetically intact source of HCMV, we cloned strain Merlin into a self-excising BAC. The Merlin BAC clone had mutations in the RL13 gene and UL128 locus that were acquired during limited replication in vitro prior to cloning. The complete wild-type HCMV gene complement was reconstructed by reference to the original clinical sample. Characterization of viruses generated from repaired BACs revealed that RL13 efficiently repressed HCMV replication in multiple cell types; moreover, RL13 mutants rapidly and reproducibly emerged in transfectants. Virus also acquired mutations in genes UL128, UL130, or UL131A, which inhibited virus growth specifically in fibroblast cells in wild-type form. We further report that RL13 encodes a highly glycosylated virion envelope protein and thus has the potential to modulate tropism. To overcome rapid emergence of mutations in genetically intact HCMV, we developed a system in which RL13 and UL131A were conditionally repressed during virus propagation. This technological advance now permits studies to be undertaken with a clonal, characterized HCMV strain containing the complete wild-type gene complement and promises to enhance the clinical relevance of fundamental research on HCMV.

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SUMMARY The antimicrobial properties of preterm and term breast milk cells were compared. They were similar in cell numbers and in the capacity to phagocytose and kill staphylococci. Interferon production on endotoxin challenge appeared to be higher in preterm cells. The antibacterial activity of breast milk cells was retained after storage at 40C for 24-48 hours. Cell numbers were unaffected by passage through a standard oral paediatric feeding set.Recent studies have reported higher concentrations of protein, sodium, and chloride in preterm than in term breast milk.1-3 These nutritional differences are thought to be of advantage to the rapidly growing infant. Less is known about the antimicrobial properties of preterm milk. Gross et al.4 recently found that preterm milk had higher concentrations of IgA. Narayanan et al.5 in a prospective controlled trial found a lower incidence of systemic infections in low birthweight infants fed their mothers' milk. In this study we examined the in vitro antimicrobial properties of preterm breast milk cells. Patients and methodsPatients. Paired samples of term and preterm breast milk were obtained from lactating mothers using an Egnell breast pump. All samples were early morning collections made at the beginning of feeds. The gestational age of the preterm infants was 27-34 (mean 31 7) weeks, their birthweights being 700-1900 (mean Duplicate mixtures of equal volumes (250 ml) of bacteria and leucocyte suspension were made in screw-topped plastic tubes and then incubated for 30 minutes at 37°C in a shaking water bath. The tubes were centrifuged at 500 g for 5 minutes and the cell pellets resuspended in 0 5 ml MEM/FCS with 100 U/ml penicillin to kill extracellular bacteria. The cells in one of the duplicate tubes were washed twice with PBS and then lysed with water to release the intracellular bacteria. The number of intracellular bacteria (= a) was then determined by the Miles-Misra method. The other tube was treated similarly after a further 2 hours' incubation at 37°C; this gives the number of bacteria (= b) surviving within the cells. The percentage of bacteria phagocytosed was calculated from the formula 100 a/c where c = the original number of bacteria.An indication of the bactericidal capacity of the leucocytes was made by calculating the percentage of the phagocytosed bacteria which were killed during the 2-hour incubation (= 100 (a-b)/a).Interferon assays. Leucocytes, suspended at 2-5 x 106/ml in MEM/FCS were incubated overnight at 37°C in the presence of 1 Ftg/ml endotoxin (lipopolysaccharide B from Escherichia coli 026-B6, Difco). The supernatants were collected, centrifuged 198 on 10 May 2018 by guest. Protected by copyright.

Serum from rabbits with BCG/endotoxin-induced shock is growth inhibitory or cytotoxic to a range ot tumour cell lines. The active component, tumour-necrosis factor (TNF), has been purified 1000-fold by sequential salt precipitation, ion-exchange chromatography and gel-filtration. TNF had a mol. wt of 67,000 on gradient PAGE and 39,000 on Ultrogel AcA44 gel-filtration. The isoelectric point was pH 5.1-5.2. TNF was susceptible to the proteolytic enzyme pronase, but resistant to trypsin or papain. On isopycnic ultracentrifugation it had a buoyant density of 1.27, confirming that it is protein in nature, with little or no carbohydrate. This is also suggested by its failure to bind to a range of lectins. Images Fig. 3