H. Kerzel Andersen

A PROSPECTIVE STUDY ON THE INCIDENCE AND SIGNIFICANCE OF CONGENITAL CYTOMEGALOVIRUS INFECTION

Abstract. Screening of 3060 neonates for congenital cytomegalovirus (CMV) infection by virus excretion in the urine showed an overall incidence of 0.4%. The incidence was about 1 % for mothers between 16 and 25 years and only 0.2% for mothers between 25 and 35. No mothers over 35 years of age gave birth to congenitally infected infants. The percentage of women in the child‐bearing age susceptible to CMV infection was estimated by the absence of CMV complement‐fixing antibodies in cord sera and ranged from 48% to 33% with increasing age. None of the infected infants showed obvious signs of congenital CMV infection at birth. At follow‐up, two infants showed slight, but transient symptoms compatible with a foetal infection; a pair of premature twins exhibited retarded physical and psychomotor development, but this could just as well be ascribed to the prematurity itself. None of the infants had detectable CMV‐IgM antibodies in cord sera, but a trend towards elevated total IgM concentration in cord sera and elevated virus excretion titres appeared in the infants with symptoms. With the very low incidence and no signs of sensomotor sequelae the preliminary conclusion is that foetal CMV infection in our population by no means has a significance to deserve routine screening or a vaccination programme.

Parathyroid glands, calcium, and vitamin D in experimental fluorosis in pigs

SummaryThe purpose of this study was to determine whether total serum calcium, parathyroid gland structure, and/or levels of parathyroid hormone, 1,25 and 24,25 DHCC, are altered in pigs with dental and skeletal fluorosis. Eight experimental animals receiving 2 mg F−/kg b.w. per day from age 8–14 months were compared with eight controls. Concentrations of plasma fluoride and total plasma calcium were assessed at intervals throughout the experiment and during a 48 hour period at day 110–111 of the experiment. At the same time, concentrations of immunoreactive parathyroid hormone were measured using a homologous labeled antibody for porcine hormone, and a radioimmunoassay was used to assess concentrations of 1,25 DHCC and 24,25 DHCC. Parathyroid tissue volumes were assessed at the end of the experiment by quantitative histology using volumetry and point counting. Plasma fluoride increased from 0.0007±0.0001 mmol/liter to 0.0127±0.002 mmol/liter in pigs receiving fluoride. In spite of this increase, total plasma calcium remained the same throughout the experiment. Volumes of parathyroid tissue, and levels of circulating parathyroid hormone 1,25 DHCC and 24,25 DHCC, were not significantly changed. It was therefore concluded that disturbance of calcium homeostasis is not an obligatory finding in dental and skeletal fluorosis and consequently does not play an essential role in the pathogenesis of these hard tissue lesions.

Serologic differentiation of human cytomegalovirus strains using rabbit hyperimmune sera

I t is generally accepted that the different human strains of cytomegaloviruses (CMV) to a wide extent share complement-fixing antigens (6, 8) whereas there is less homogeneity in neutralization tests (10, ll) . Serologic differentiation of human C1V[V strains by cross neutralization tests (NT) have been complicated for more than a decade because it proved impossible to produce potent hyperimmune sera in rabbits (8). Recently, however M-4NT~JXI~VI (9) succeeded in producing hyperimmune sera against strain Ad169 although with relatively low NT titers. Attempts at serologic differentiation of human strains of CMV have for this reason up till now been undertaken to a minor extent only, using convalescent sera (11), hyperimmune sera from monkeys (10) or guinea pigs (7), and they have not provided convincing evidence for the existence of clearly different serotypes of human CMV. In this laboratory a CMV strain (T27) was isolated which on the basis of preliminary titration experiments with the patients own serum appeared to differ antigenically from the l~boratory strain Ad 169. In order to perform a more extensive serologic comparison between these two strains an attempt was made to produce antisera by the immunization of rabbits.

CYTOMEGALOVIRUS INFECTION AMONG INFANTS ADMITTED TO A PAEDIATRIC DEPARTMENT

Infection with cytomegalovirus (CMV) is common in infants and children. The infection can be demonstrated by isolation of virus from urine and sputum, and by estimation of seroantibodies. Both virus excretion and seroantibodies are known to persist for long periods of time after primary infection with CMV. Starr & Gold (16) have reported the finding of CMV-uria at birth in 1.57% of 507 unselected neonates, whereas Stern (17) in a study of 118 newborns and 136 hospitalized children aged 2 months to 4 years found CMV-uria in 3% and l o % , respectively. Complement fixing (CF) antibodies are found with quite varying frequency in children from the age of 4 to 2-5 years; thus, by Stern & Elek (18) and Hanshaw (10) in about 406 ; by Rowe et al. (15), Carlstrom (6), VBczi et al. (19), and Baerlocher et al. (5 ) , in about 20% and by Chiba et al. (8) in about 60%. The relatively higher frequency of seropositivity among infants less than 6 months of age has been ascribed to the presence of maternal antibodies during the first months of life by the above-mentioned authors. This report concerns the results of a study of CMV infections in infants less than 1 year of age admitted to a paediatric department during the course of 12 months.

The influence of complement on cytomegalovirus neutralization by antibodies

The influence of complement on the neutralization of cytomegalovirus by antibodies has been studied using human sera and antisera prepared in rabbits with various strains of cytomegalovirus (Ad 169, C 87, Davis andT 27). Examination of patient sera showed that addition of 2 per cent complement had only a weak and non-significant enhancing effect on neutralization, even by sera taken early in the course of infection. It proved possible to produce specific antisera with high titres in rabbits with the four strains of cytomegalovirus. It was found that addition of 2 per cent complement had an enhancing effect on neutralization of cytomegalovirus by hyperimmune sera. Concerning the Ad 169 and C 87 antisera the effect of complement was essentially limited to early antisera, whereas the effect was especially pronounced on late Davis and T 27 antisera. The late Ad 169 and C 87 sera did not require complement, whereas all late Davis and T 27 antisera were complement-requiring. These differences, which most likely are bound to whether the anti bodies are γG or γM antibodies make it important to use complement in cross-neutralization experiments with hyperimmune sera.

Studies of human cytomegalovirus strain variations by kinetic neutralization tests

Three laboratory strains of human cytomegalovirus (CMV) (Davis, Ad 169 and C 87) and one local strain (T 27) have been compared antigenically using 4 human sera and rabbit hyperimmune-sera to each strain. The human sera and the Ad 169 and C 87 hyperimmune-sera contained complement (C′)-independent antibodies whereas the antibodies produced with strain Davis and T 27 were C′-dependent. The results of the study indicate that rabbit-CMV-hyperimmune sera, whether C′-dependent or not, as well as human sera can be used to demonstrate cross reactions among CMV strains as well as antigenic variations. As all sera neutralized all strains, but mostly with various rates, it is concluded that the 4 strains tested share neutralizing antigens. None of the strains, however, seemed to be antigenically identical. Strain T 27 appeared to be the most distinct from the rest, whereas major serologic differences were not seen between strains Davis and Ad 169. For that reason it does not seem reasonable to retain the terms Types 1 and 2 for strain Davis and Ad 169. About 200 human sera were screened for neutralizing antibodies using all strains or the 2 most diverse strains (Ad 169 and T 27). The individual sera in low dilution were able to neutralize either all strains or none. On the basis of the kinetic studies strain Ad 169 was found broadly reactive and thus well suited for determination of neutralizing antibodies in human sera.

Studies on human cytomegalovirus strain variations by membrane-fluorescence

I t is well documen ted t h a t the h u m a n cytomegalovi ruses (CMV) share ant igens to a wide extent . Using sensi t ive serological methods , ant igenic differences can, however , be demons t r a t ed among var ious s t ra ins (1--3 , 6). A new CMV an t igen loca ted on the cell m e m b r a n e of CMV-infected h u m a n f ibroblas ts has r ecen t ly been de tec ted (5). The a im of the p resen t s t u d y was to inves t iga te whe ther m e m b r a n e ant igens induced b y 3 l a b o r a t o r y s t ra ins of CMV possess s t ra in specifici ty. The CMV st ra ins Ad169, Davis and C87 toge ther wi th the techniques for p roduc ing CMV h y p e r i m m u n e sera in r abb i t s wi th these s t ra ins have been described p rev ious ly (1, 2). F i b r o b l a s t an t ibodies were r emoved b y absorb ing the h y p e r i m m u n e sera on washed, noninfec ted h u m a n I ibroblas t s (8 • 107 cells/0.5 ml serum, d i lu ted 1:5) for ] hour a t room t e m p e r a t u r e and then a t 4 ~ C overnight . I n add i t i on the sera were abso rbed twice wi th the same a m o u n t of f reezethawed, d i s rup ted cells for 1 hour a t room t e m p e r a t u r e and then a t 4 ~ C overnight . F ina l l y the sera were spun a t 10,000 r. p. m. for 30 minu tes and s tored a t 2 0 ~ C. All sera were s tud ied in a d i lu t ion of 1:10 in the membrane-f luorescence tes t aga ins t noninfec ted f ibroblas ts and found negat ive . Coversl ip cul tures were p repa red in 6 cm Pe t r i dishes seeded wi th 7.5 • 105 h u m a n f ibroblas ts and incuba ted a t 37 ~ C for 2 days . They were inocula ted with 1 5 p. f. u. virus/cel l and incuba ted for 4 days , a f te r which 80-90 per cent of the cells exh ib i t ed ey~opathic changes. The indirect membraneimmuno-f luorescence tes t w~s pe r fo rmed on the washed, nonf ixed coversl ip cul tures b y t r ea t ing wi th d i lu ted h y p e r i m m u n e serum for 30 minutes a t room t empera tu re . ~k[ter washing thr ice wi th buffered saline the p repa ra t i ons were covered for the same per iod of t ime wi th F I T C c o n j u g a t e d a n t i r a b b i t I gG globul in d i lu ted 1:60. Af te r th ree fur ther washes the p repa ra t ions were m o u n t e d in buffered glycerol .

The Influence of Cyclosporin on Cytomegalovirus-Related Kidney Graft Survival

In 1981, we reported that one-year actuarial kidney graft survival was much better in recipients with no evidence of cytomegalovirus (CMV) infection than in recipients with primary CMV infection (1). At that time standard post-transplant therapy consisted of prednisone and azathioprine. Four-and-a-half-years ago azathioprine was replaced with cyclosporin. The present paper concerns graft survival in relation to CMV in these patients.

Donor Cytomegalovirus Immunity is a Risk Factor for Chronic Graft-Versus-Host Disease (GvHD) After Allogeneic Bone Marrow Transplantation

Chronic Graft-versus-host disease (GvHD) develops in approximately 30% of patients after allogenic bone marrow transplantation, presumably mediated by an immunologic reaction of transplanted donor cells against recipient antigens. Recipient age and previous acute GvHD have been found to predispose for this complication1. More recently, it has been reported that the onset of chronic GvHD frequently is preceded by a cytomegalovirus (CMV) infection2. We hypothezised that an immunologic reaction of donor cells against CMV infected recipient cells could be involved in the pathogenesis of chronic GvHD. We therefore decided to analyse the effect of donor pretransplant CMV immunity on the incidence of chronic GvHD.