Human cytotrophoblastic cells absorb the NK blocking activity of monoclonal BA11.

PROBLEM: R80K is a polymorphic alloantigeneic protein present on human placental trophoblast and on paternal B lymphocytes and monocytes. This protein, unlike the former candidate TLX antigen, stimulates a protective maternal immune response in vivo. A murine monoclonal BA11 antibody, directed against R80K, prevents abortion in three murine pregnancy-failure models and inhibits human and murine NK activity. We attempted to define the target of BA11 in the human NK assay system. METHODS: A CELISA method was used to detect R80K antigen on the surface of different cells using the BA11 antibody. The effect, on human peripheral blood NK activity against K562, by BA11 before and after absorption by different cells, including the K562 target, was determined. RESULTS: R80K was detected on term placental syncytio and cytotrophoblast and on BeWo cells, by CELISA. BA11 suppressed NK lysis of K562 cell sin a dose-dependent manner. Absorption of the BA11 by BeWo and by cytotrophoblastic cells significantly decreased the NK-inhibitory activity. There was minimal absorption by K562 and BA11-pretreateed K562 cells remained susceptible to NK lysis. By contrast, BA11-pretreated peripheral blood cells lost all NK activity. CONCLUSIONS: The inhibition of NK killing of K562 cells by BA11 is more complex than simple masking of a trophoblast cell-associated molecule in K562 necessary for recognition in NK cells.

Immunosuppressive properties of monoclonal antibodies and human polyclonal alloantibodies to the R80K protein of trophoblast.

PROBLEM: The R80K protein on human trophoblast is antigenically polymorphic, and in all placentae of successful pregnancies, the protein is covered by maternal alloantibody. Alloantibody eluted from human placenta has been shown to inhibit killing by human NK cells. Do those antibodies to R80K that inhibit NK killing also affect the murine abortion models? METHODS: We made three murine monoclonal antibodies to conserved epitopes, on human R80K, all of which also reacted with the homologous murine molecule. One antibody only, BA11, suppressed NK cytotoxicity to K562 and of mouse spleen NK cells to murine trophoblast. All three were tested in mouse models of abortion: the CBA x DBA/2 model with a high resorption rate of F1 embryos compared with the parental strains, an endotoxin induced abortion/resorption model and a third model in which the pregnant mouse is subject to sonic stress. CONCLUSION: Those IgG antibodies eluted from microvesicles which bound to K562, and one of the three monoclonals, BA11, inhibited NK killing. The antibodies react with the murine molecule, and BA11 inhibited abortion in all three mouse abortion models. This reinforces the thesis that interference with NK killing can influence abortion/resorption in mice, and the BA11 antibody may effect similar results in analogous human situations.

Psycho-neuro-cytokine/endocrine pathways in immunoregulation during pregnancy.

PROBLEM: Some mammalian pregnancy failure is thought to occur by immunological or immunologically modifiable mechanisms. The original model wherein spontaneous abortion was proposed to represent rejection of the conceptus as an allograft has been supplanted by a model of maternal paraimmunological natural effector cell toxicity to fetal trophoblast more closely related to tumor rejection. The problem is to integrate current information concerning the role of immunological, paraimmunological, endocrinological, and stress-triggered neural factors that determine whether or not abortion will occur. METHODS: Review of existing data. RESULTS: An integrated model is proposed. CONCLUSION: Immunological factors play an important role in abortion processes and prevention of abortions. The existence of abortogenic mechanisms and their regulation appears to be based upon optimizing survival of the species. Two new conceptual models provide a useful framework for further investigation of human pregnancy failure and its treatment.

Anomalous inheritance of a paternally derived trophoblast antigen.

PROBLEM: Recurrent spontaneous abortion occurs in 1 in 500 random matings and usually results in abortion of all pregnancies. If absence of antibody to a paternally derived antigen caused abortion, the woman would be expected to make antibody to the other paternal antigen and abort only half her pregnancies. METHODS: Microvesicles were prepared from equine placentae. Acid-eluted IgG antibody was eluted from the polymorphic R80K antigen and used to type the residual R80K antigen on vesicles or on peripheral blood leucocytes. RESULTS: In several equine sibships all the half-sibs had the same paternal R80K alloantigen. In the extended horse family descended from the stallion Nearco, three allotypes were found. The allele present was usually the grandpaternal one, but exceptions are seen. Whichever allele is transmitted all the progeny have the same alloantigen (probability of this occurring by chance = 2(-45)). CONCLUSION: Because only one paternal allotype is present in all progeny, lack of antibody to the R80K antigen would result in loss of all pregnancies, not one half.

An 80-kDa syncytiotrophoblast alloantigen bound to maternal alloantibody in term placenta.

PROBLEM: We have shown that most of the IgG present on term syncytiotrophoblast, membrane, microvesicles is bound to an 80 kDa protein antigen (R80K). METHODS: Microvesicles were prepared from term human placenta, and the IgG eluted at pH3. RESULTS: When IgG antibody was eluted at pH3 and reacted with acid-treated vesicles of other placentae, the alloantibody always bound to the preparation from which it was obtained, but only to about 10% of acid-treated preparations from other placentae. A similar polymorphic protein found in association with IgG antibody was found in term horse placentae. Cross-reactivity of the antibodies between species was not found. Using binding of labelled antibody, complement dependent cytotoxicity and FACS two-color analysis, the human polymorphic antigen was present on peripheral blood monocytes and B-lymphocytes. The R80k antigen on intact microvesicles was resistant to trypsin, but after acid elution of IgG, trypsin released a soluble 50 kDa fragment which reacted with the acid-eluted IgG antibody. CONCLUSION: The presence of antibodies to R80K in all term placentae studied, including first pregnancies, suggests that development of this alloantibody may be a normal requirement for successful pregnancy.

On genetic and environmental factors in Menière's disease.

The etiology of Menière's disease (MD) remains obscure. Previous studies have shown a highly significant association between sporadic MD and one of the human leukocyte antigen, HLA-C genotypes, whereas disease activity has been related to the detection of enterovirus-specific viral protein (VP1) in the peripheral circulation. This present research extends the HLA association of sporadic cases to the study of families with more than one living member with unequivocal MD. Since the sporadic HLA associations point to chromosome 6 being a candidate region of a possible MD mutation, this area of the human genome has been investigated first; DNA suitable for study by other markers has been stored. The presence or absence of VP1 in the familial MD patients has been measured and related to disease activity at the time of sample collection. The association, in both sporadic and familial cases, of MD and partial HLA class I haplotypes points to a likely MD locus lying between the HLA-C and HLA-A loci on the short arm of chromosome 6. The significant relation between disease activity and circulating VP1 has been confirmed. It is likely that the predisposition to familial MD is attributable to a mutation on chromosome 6, which has been designated M1.

Immunology of postviral fatigue syndrome.

Postviral fatigue syndrome is associated with persistent infection by a virus. The patient with the condition has failed to eliminate the virus in the usual time. There is little evidence of a deficient immune response by the patient as the explanation for the viral persistence, and it must be assumed that most of the explanation lies in down-regulation of virus expression in infected cells. The general symptomatology of postinfectious syndromes may be mediated by cytokines liberated as part of the infection. Part of the syndrome may also be due to local effects of virus infection in muscles or the central nervous system (CNS).

Detection of enterovirus specific RNA sequences in muscle biopsy specimens from patients with adult onset myositis.

A subgenomic cDNA probe with broad specificity for a range of enteroviruses was used to test by an in situ hybridisation technique for the presence of enterovirus specific genomic sequences in muscle biopsy samples obtained from patients with chronic adult myositis. Virus specific RNA sequences were detected in 6/13 (46%) patients with idiopathic polymyositis or dermatomyositis. Control samples obtained from an equal number of patients suffering from other muscle disorders were negative. A monoclonal antibody specific for an enterovirus group was used to probe for viral antigens by indirect immunoperoxidase staining; all biopsy samples from test and control groups were negative.

Persistence of hepatitis A virus in fulminant hepatitis and after liver transplantation.

A peroxidase-labelled, specific mouse monoclonal antibody to hepatitis A virus (HAV) and an in situ hybridization technique (streptavidin-biotin-horseradish peroxidase reaction) with an HAV-specific cDNA probe (recombinant plasmid pAWHA comprising 1.8 kb of the HAV-specific cDNA, located toward the 3' end of the genome) were used to detect HAV in liver tissues in two patients with fulminant viral hepatitis type A treated by liver transplantation after a protracted (day 40: case 1) and relapsing (day 60: case 2) clinical course. HAV antigens and HAV-specific genomic sequences were detected in the hepatectomy tissues and in serial biopsies of the liver grafts through to final follow-up at 2 months (case 2) or death at 7 months after re-grafting for chronic rejection (case 1). In the fulminant liver parenchyma, numerous degenerating and some surviving hepatocytes were positive and randomly scattered. The immunoperoxidase staining was predominantly cytoplasmic and often granular. The localization of the cDNA probe was predominantly nuclear/perinuclear but was occasionally cytoplasmic. High-titre IgM-anti-HAV antibodies persisted until death (case 1) or resolution (5 months) of an acute hepatitis (case 2), which occurred at 2 months, accompanied by HAV antigen (ELISA), in stool. Intact replicating virus particles must have been present in one or more sites in each case, including extrahepatic locations, with a viraemia as the most likely explanation for subsequent reinfection of the grafts.

Postviral syndrome--how can a diagnosis be made? A study of patients undergoing a Monospot test.

Eighty-nine of 150 patients having a Monospot test filled out a questionnaire about their illness, and the General Health Questionnaire. They completed a follow-up questionnaire 6 months later. Twelve (8%) had a positive Monospot. Twenty-eight of 83 serum samples tested (34%) were positive for VP1 enteroviral antigen. Forty of the patients had a self limiting illness, 13 had a definite diagnosis (excepting glandular fever), 14 had a possible postviral syndrome, 10 had recurrent sore throats/flu, and 12 had a chronic non-specific illness. Patients with a specific diagnosis were less likely to complain of aching muscles/joints, sore throat, tiredness or loss of concentration. Their GHQ scores were lower, although this just failed to reach significance (P = 0.08), and they scored significantly lower on the somatic symptoms subscale (P = 0.022). Overall 72% scored above the GHQ threshold for 'psychological caseness' which is higher than in other studies. Sixty-five per cent of the sample questioned at 6 months felt that their illness started with a viral infection. The methodological problems involved in making a diagnosis of postviral syndrome are discussed.

Histologic and immunologic study of uterine biopsy tissue of women with incipient abortion.

Biopsy specimens taken from the region of the placental bed were examined for the presence of phloxinophilic granulated mononuclear cells in women with a history of recurrent miscarriage and who would eventually miscarry a current pregnancy. They were compared with biopsy specimens from women with intact pregnancies presenting for elective termination of pregnancy and those with "missed abortion." Cells with large cytoplasmic granules (greater than or equal to 1 micron) were abundant in the group of ongoing pregnancies whereas cells with smaller granules (less than 1 micron) that were similar to large granular lymphocytes were more abundant relative to cells with large granules in the biopsy specimens from failing pregnancies. Immunosuppressive activity was tested in the supernatants of cultured biopsy samples of each group and found to be significantly lower in the incipient miscarriage group. These findings could represent alterations associated with the process of miscarriage, such as inflammation, or there may be deficient suppressor cell activity at the fetomaternal interface as the reason for "rejection" of the early embryo.

Detection of enterovirus RNA in experimentally infected mice by molecular hybridisation: specificity of subgenomic probes in quantitative slot blot and in situ hybridisation.

Subgenomic cDNA clones representing defined regions of the genome of Coxsackie B3 virus were used as hybridisation probes to detect RNA of various enteroviruses in cell culture and mouse model systems. Radiolabelled probes were used in slot blots to quantitate the RNA in samples; biotinylated probes were used to localise virus RNA at the cellular level by in situ hybridisation with monolayers of infected cells or thin sections of tissue samples. Probes derived from the 5' or 3' terminal regions of Coxsackie virus RNA, which are highly conserved in the genus Enterovirus, detected RNA of various serotypes in infected cell cultures, but failed to hybridise with hepatitis A virus (HAV) RNA. Although HAV is clearly a Picornavirus, our data support the view that its taxonomic position within the enteroviruses should be reconsidered. The biotinylated probes were also used to detect in situ virus RNA in paraffin-embedded tissue samples from experimental mouse models of Coxsackie B3 virus-induced myocarditis or Coxsackie B1 virus-induced myositis. Since the integrity of the tissues was preserved during the process, and viral RNA was localised in the affected muscle fibres, this has enabled us unequivocally to relate the infecting virus to the underlying tissue injury.

Immunology of early pregnancy.

The results of immunization of women with recurrent spontaneous abortion with paternal lymphocytes are described. The women we have treated were those with more than two spontaneous abortions with the same partner, who did not have evidence of genetic or other causes of recurrent abortion. Only women with recurrent abortion and no more than one live child were considered suitable for treatment. The success of treatment before pregnancy was 77%, similar to that found in our original double-blind controlled trial of immunization. The failures of treatment were found to cluster in those women who did not make detectable cytotoxic antibody after treatment, and who did not become pregnant within 90 days. Those women who did develop antipaternal antibody after immunization showed a protective response with no significant drop in efficiency in the year after immunization. A further group of women are described who were treated in the early part of pregnancy. The success rate was very high when the treatment was given within 40 days of the last period, and was less effective the later in pregnancy it was given. Birthweight, gestational age and malformation rate of the babies born after treatment were not different from those found in the normal population.

Chronic enterovirus infection in patients with postviral fatigue syndrome.

76 patients with the postviral fatigue syndrome (PVFS) and 30 matched controls were investigated. Virus isolation was attempted from concentrated faecal samples by direct culture and after acid dissociation of virus from antibody. Positive cultures of enteroviruses were obtained from 17 (22%) patients and 2 (7%) controls. An enterovirus-group-specific monoclonal antibody, 5-D8/1, directed against the VP1 polypeptide, was used to detect enteroviral antigen in the circulation, either free or complexed with antibody. VP1 antigen was detected in the serum of 44 (51%) of a further group of 87 PVFS patients. The number of patients positive for VP1 antigen was greater (42/44) when IgM complexes were detectable than when they were not (2/23). 1 year later, the 17 patients of the first group of 76 with positive cultures were again studied. The same virus was again isolated from 5 (29%), 13 (76%) had detectable IgM responses to enteroviruses, and 9 (53%) were positive for VP1 antigen in the serum. These results show that chronic infection with enteroviruses occurs in many PVFS patients and that detection of enterovirus antigen in the serum is a sensitive and satisfactory method for investigating infection in these patients.