Linda L. Pifer

Propagation of Pneumocystis Carinii In Vitro

Summary: Pneumocystis carinii was propagated in vitro with primary embryonic chick epithelial lung (CEL) cells. Viability and growth of the organism were demonstrated by direct observation of the reproductive cycle in the Sykes-Moore chamber, serial passage with an increase in the number of mature cyst forms, the cytopathic effect of the organism on cell culture, and inhibition of growth of the organism by specific antiserum and pentamidine isethionate. Attempts to culture P. carinii indefinitely were not successful. However, cyst forms derived from murine and human sources increased 100-fold and 10-fold, respectively, during CEL cell culture passages. Serial passage of trophozoites alone resulted in the development of typical CPE and a maximum number of 2.8 103 cyst forms. Autoradiographic methods demonstrated active DNA, RNA, and protein synthesis within the cyst and suggest that metabolic interaction between the host cells and the organisms occurred. The nature of the attachment of P. carinii to the host CEL cell was clearly discernible by scanning electron microscopy (SEM).In the reproductive cycle a vegetative cell (designated “trophozoite”) attached by tubular expansions to the host CEL cell, probably for the transport of essential nutrients, and then detached without entering the cell. Sporozoites developed within the detached young cyst, reaching a maximum number of eight within the mature cyst. Excystment occurred through single or multiple sites in the cyst wall, after which the released trophozoite attached to a new host cell.Speculation: The in vitro propagation of P. carinii provides a basis for further studies to characterize the biologic and biochemical features of this organism and a source of antigen for the development of serologie tests for specific antibody by which the epidemiology and spectrum of clinical disease can be delineated more clearly.

Gamma irradiation of HIV-1

The potential for transmission of human immunodeficiency virus (HIV) type 1 has created serious concern for the continued clinical use of bone and soft‐tissue allografts. Tissue banks have employed 1.5–2.5 Mrad for sterilization of bone and tendon allografts, which, according to the current literature, approaches the level at which the tissue quality is adversely affected for implantation. Our working hypothesis was that gamma irradiation at increasing doses can proportionately inactivate HIV type 1. The objective of this study was to inactivate HIV type 1 by irradiation, as determined by its capacity to infect human T‐lymphocytes and established cell lines in vitro. The replicative competence of HIV type 1 was also assessed by the presence of reverse transcriptase activity, enzyme‐linked immunoadsorbent assay (ELISA), immunofluorescence assays for p24 viral core antigen, and the formation of syncytia induced by HIV type 1 in the cultures inoculated with irradiated virus. The results demonstrated the presence of active viral replication in previously noninfected cells in the supernatant samples that were exposed to as much as 5.0 Mrad. The data for the 10‐Mrad sample were indeterminate due to cellular damage. These data suggest that gamma irradiation (1.5–2.5 Mrad) does not constitute a virucidal dose for HIV type 1. Current technologies for screening have greatly improved, and the surgeon should rely on tissue bank screening procedures and other methods of preparation rather than sterilization by gamma radiation techniques in choosing allograft material.

Pneumonia associated with infection with pneumocystis, respiratory syncytial virus, chlamydia, mycoplasma, and cytomegalovirus in children in Papua New Guinea.

Paired serum samples were collected from 94 children with pneumonia admitted to Goroka Hospital, Papua New Guinea. All but three of the children were aged 1-24 months. Only nine children were malnourished, with weight for age less than 70% of the Harvard median (three had weight for age less than 60% of the Harvard median). Pneumocystis carinii antigen was detected in the serum of 23 children. Twenty two children had serological evidence of recent infection with respiratory syncytial virus. Five children were probably infected with Chlamydia trachomatis at the time of the study, and there was less convincing serological evidence of current infection in a further 11 children. Five children showed a fourfold rise in antibody to Mycoplasma pneumoniae. Although only one child showed a fourfold rise in antibody to cytomegalovirus, 86 children had this antibody. No child showed a fourfold rise in antibody to Ureaplasma urealyticum or Legionella pneumophila. P carinii, respiratory syncytial virus, C trachomatis, M pneumoniae, and cytomegalovirus may be important causes of pneumonia in children in developing countries.

EFFICACY OF INTRAVENOUS GAMMAGLOBULIN IN THE TREATMENT OF PNEUMOCYSTIS CARINII PNEUMONIA IN THE RAT MODEL

The efficacy of commercially-prepared IV infusions of pooled human immunoglohulin G (IVIG) was evaluated in the treatment of Pneumocystis carinii pneumonia (PCP) in the immunosuppressed rat model to ascertain whether such treatment might prove feasible in the managment of pediatric subjects with PCP. Forty-eight rats were divided into six groups: (1) controls (C), (2) controls given IV human gammaglohulin infusions (C-I), (3) immunosuppressed rats given short-term cortisone (STC), (4) immunosuppressed rats given short-term cortisone and IV human gammaglobulin infusions (STC-I), (5) immunosuppressed rats given long-term cortisone (LTC), and (6) immunosupressed rats given long-term cortisone and IV human gammaglobulin infusions (LTC-I). An enzyme-linked immunosorbant assay (ELISA) was employed to monitor PC-specific endogenous and exogenously administered IgG throughout the test period and toluidine hlue ‘0’ stains were performed to determine the degree of P. carinii infection in rat lung. Lung infection analysis showed statistical equivalence in STC, STC-I, and LTC-I. However, LTC-I lungs contained significantly fewer (P<.05) cysts than did LTC rats not treated with IVIG. IVIG appeared efficacious in reducing the number of cysts in the lungs of immunosuppressed rats with long-term cortisone. This study suggests a significant role for humoral immune defenses in combating PCP and warrants controlled studies in the use of IVIG in the pediatric. patient with PCP.

1153 PNEUMOCYSTIS CARINII ANTIGEN AND ANTIBODY IN CHILDREN WITH PNEUMONIA

Serologic profiles of Pneumocystis carinii (PC) based upon incidence of antigenemia & IgG antibody (AB) titers in pediatric patients in Papua, New Guinea (NG) & Memphis, TN support the ubiquity of PC. PC IgG AB was titered by an enzyme-linked immunosorbent assay & antigenemia was detected by a counterimmunoelectrophoresis test, both developed in our laboratory. Antigen (AG) used in the ELISA test was derived from cell culture-grown PC. NG children with pneumonia (N=188) had higher anti-PC titers than U.S. controls (N=50). Those with PC AG (+) pneumonia had highest titers (geometric mean titer = 277), suggesting a specific immune response to PC. Those with PC AG (-) pneumonia (GMT=139) had higher titers than matched U.S. controls (GMT=60), perhaps reflecting more environmental exposure or differences due to socio-economic status. The extent to which PC is responsible for morbidity in multiple-agent pneumonias remains to be elucidated. Since PC is treatable, this represents a high priority goal, as the decision to treat might influence survival in critical cases. Mean AB titers were all significantly different (p=<.050). This suggests the potential usefulness of serologic methods in documenting the involvement of PC in childhood pneumonia & in defining its epidemiology. These tests may provide a means for differentiating among pediatric pneumonias due to PC, Chlamydia, & cytomegalovirus, which may be clinically indistinguishable.