Abraham Morag

PROLONGED ATYPICAL ILLNESS ASSOCIATED WITH SEROLOGICAL EVIDENCE OF PERSISTENT EPSTEIN-BARR VIRUS INFECTION

Seven patients with prolonged atypical illness were followed up for more than a year. Sera taken during that period showed significantly increased titres of IgM antibodies against the viral capsid antigen (VCA) of Epstein-Barr virus (EBV). In four of the patients antibodies to the R component of the early antigen (EA) complex of EBV were clearly detectable. Only one of these seven patients had presented with symptoms of classic infectious mononucleosis. Serological and clinical observations in these patients suggest that the prolonged atypical illness was probably the result of persistent EBV infection.

Psychological variables as predictors of rubella antibody titers and fatigue—a prospective, double blind study

When exposed to infectious pathogens, human beings manifest variability in the incidence and severity of infection. This variability may partly depend on psychological variables, which have long been thought to contribute to the predisposition, onset, and course of various physical illnesses, including infectious diseases. The objective of the study was to investigate the predictive value of several personality and other psychological variables on antibody titers and fatigue following a specific viral infection. Subjects were divided into a seronegative group (not immune prior to vaccination) (N = 60) and a seropositive group (immune prior to vaccination) (N = 180), based on antibody titers to rubella before and 10.5 weeks after vaccination with live-attenuated rubella virus. Questionnaires assessing externalizing, internalizing, self-esteem, neuroticism, and fatigue-related symptoms were administered to the subjects before vaccination. Fatigue-related symptoms were re-evaluated 10 weeks post vaccination. In the seronegative group, low titers of rubella antibodies, 10.5 post-vaccination, were predicted by high internalizing or neuroticism scores, and by low self-esteem, measured at baseline. Higher externalizing scores indirectly predicted lower titers of antibodies, via fatigue-related symptoms, measured 10 weeks post vaccination. In contrast, in the seropositive group no association was found between any of the psychological variables and antibody titers. Personality and other psychological variables can predict antibody titers to rubella vaccination, in infected individuals. The associations between the psychological variables and antibody titers are complex, and involve both direct and indirect associations. Specific psychological variables can also be used to predict levels of post-vaccination fatigue.

Role of leukocyte factors and cationic polyelectrolytes in phagocytosis of group A streptococci and Candida albicans by neutrophils, macrophages, fibroblasts and epithelial cells: modulation by anionic polyelectrolytes in relation to pathogenesis of chronic inflammation.

A variety of cationic polyelectrolytes opsonized group A streptococci andCandida albicans to phagocytosis by human polymorphonuclear leukocytes and by mouse peritoneal macrophages. The most potent opsonins for streptococci were specific antibodies supplemented with complement, nuclear histone, polylysine, polyarginine, ribonuclease, leukocyte lysates, leukocyte cationic protein and, to a lesser extent, lysozyme and myeloperoxidase. Histone, RNAse, leukocyte extracts, and platelet extracts also functioned as opsonins for phagocytosis of streptococci in the peritoneal cavity, where phagocytic indices, higher than those obtained for the in vitro phagocytosis, were obtained. Fresh serum, polylysine, polyarginine, and nuclear histone acted as good opsonins forCandida, but none of the other factors tested were active. In order for the cationic proteins and leukocyte extracts to function as opsonins, they must be present on the particle surface. These agents were poor opsonins when applied on the macrophages. Nuclear histone, polylysine, polyarginine, and fresh human serum also functioned as good opsonins for the uptake ofCandida by mouse fibroblasts. On the other hand, none of the other substances which opsonized streptococci were effective withCandida. The phagocytic capabilities of fibroblast polykaryons were much higher than those of ordinary spindle-shaped mouse fibroblasts. Histone also functioned as a good opsonic agent for the uptake ofCandida by human fibroblasts, HeLa cells, epithelial cells, monkey kidney cells, and rat heart cells. On the other hand, neither leukocyte extracts nor ribonuclease LCP or MPO functioned as opsonins for these mammalian cells.Candida, taken up by fibroblasts, were present within tight phagosomes, but no fusion of lysosomes with the phagosome occurred. A small proportion of the internalized yeast cells underwent partial plasmolysis, but little damage to the rigid cell walls was observed within 24–48 h of internalization. Phagocytosis of streptococci andCandida by macrophages and the uptake ofCandida by fibroblasts were both strongly inhibited by liquoid (polyanethole sulfonic acid sodium salt). This anionic polyelectrolyte also markedly inhibited the release ofN-acetylglucosaminidase from macrophages without affecting cell viability (LDH release). Hyaluronic acid, DNA, and dextran sulfate markedly inhibited the uptake of histone-coated particles by macrophages. On the other hand, hyaluronic acid and DNA enhanced the uptake ofCandida by fibroblasts. The effect of these anionic polyelectrolytes on phagocytosis of serum-opsonized particles by macrophages was not consistent. While in some experiments it blocked phagocytosis, in others it either had no effect or even enhanced the uptake of the particles. Phagocytosis of microorganisms by “nonprofessional” phagocytes like fibroblasts and the paucity in these cells of hydrolases capable of breaking down microbial cell wall components may contribute to the persistence of non-biodegradable components of bacteria in tissues and to the perpetuation of chronic inflammatory sequellae. Cationic polyelectrolytes may also prove important as “helper” opsonins and as agents capable of enhancing the penetration into cells of both viable and nonviable particles, genetic material, and drugs.A variety of cationic polyelectrolytes opsonized group A streptococci andCandida albicans to phagocytosis by human polymorphonuclear leukocytes and by mouse peritoneal macrophages. The most potent opsonins for streptococci were specific antibodies supplemented with complement, nuclear histone, polylysine, polyarginine, ribonuclease, leukocyte lysates, leukocyte cationic protein and, to a lesser extent, lysozyme and myeloperoxidase. Histone, RNAse, leukocyte extracts, and platelet extracts also functioned as opsonins for phagocytosis of streptococci in the peritoneal cavity, where phagocytic indices, higher than those obtained for the in vitro phagocytosis, were obtained. Fresh serum, polylysine, polyarginine, and nuclear histone acted as good opsonins forCandida, but none of the other factors tested were active. In order for the cationic proteins and leukocyte extracts to function as opsonins, they must be present on the particle surface. These agents were poor opsonins when applied on the macrophages. Nuclear histone, polylysine, polyarginine, and fresh human serum also functioned as good opsonins for the uptake ofCandida by mouse fibroblasts. On the other hand, none of the other substances which opsonized streptococci were effective withCandida. The phagocytic capabilities of fibroblast polykaryons were much higher than those of ordinary spindle-shaped mouse fibroblasts. Histone also functioned as a good opsonic agent for the uptake ofCandida by human fibroblasts, HeLa cells, epithelial cells, monkey kidney cells, and rat heart cells. On the other hand, neither leukocyte extracts nor ribonuclease LCP or MPO functioned as opsonins for these mammalian cells.Candida, taken up by fibroblasts, were present within tight phagosomes, but no fusion of lysosomes with the phagosome occurred. A small proportion of the internalized yeast cells underwent partial plasmolysis, but little damage to the rigid cell walls was observed within 24–48 h of internalization. Phagocytosis of streptococci andCandida by macrophages and the uptake ofCandida by fibroblasts were both strongly inhibited by liquoid (polyanethole sulfonic acid sodium salt). This anionic polyelectrolyte also markedly inhibited the release ofN-acetylglucosaminidase from macrophages without affecting cell viability (LDH release). Hyaluronic acid, DNA, and dextran sulfate markedly inhibited the uptake of histone-coated particles by macrophages. On the other hand, hyaluronic acid and DNA enhanced the uptake ofCandida by fibroblasts. The effect of these anionic polyelectrolytes on phagocytosis of serum-opsonized particles by macrophages was not consistent. While in some experiments it blocked phagocytosis, in others it either had no effect or even enhanced the uptake of the particles. Phagocytosis of microorganisms by “nonprofessional” phagocytes like fibroblasts and the paucity in these cells of hydrolases capable of breaking down microbial cell wall components may contribute to the persistence of non-biodegradable components of bacteria in tissues and to the perpetuation of chronic inflammatory sequellae. Cationic polyelectrolytes may also prove important as “helper” opsonins and as agents capable of enhancing the penetration into cells of both viable and nonviable particles, genetic material, and drugs.

INCREASED (2'-5')-OLIGO-A SYNTHETASE ACTIVITY IN PATIENTS WITH PROLONGED ILLNESS ASSOCIATED WITH SEROLOGICAL EVIDENCE OF PERSISTENT EPSTEIN-BARR VIRUS INFECTION

initial finding of raised anti-VCA IgM antibody. Very high levels of enzyme activity, similar to those observed in acute infectious mononucleosis,’ were found in all the patients tested (see table). Interferons (IFNs) are produced in response to viral infections. Among the biochemical changes caused in cells by IFNs is the induction of the enzyme oligo-A synthetase,2 thus an increased activity of this enzyme indicates that the host cells have been exposed to IFN and are responding to it. Increased levels of this enzyme have been observed during acute viral infections, collagen diseases, and EBV-related malignancies. In mice injected intraperitoneally with vesicular stomatitis virus and Sindbis virus, (2’-5’)oligo A levels return to normal within 1-4 weeks.3 After acute infectious mononucleosis, activity of the enzyme seems to decline to normal levels over several weeks. Where the illness was prolonged, increased levels of enzyme activity were found many months after the onset of signs and symptoms. Enzyme activity was measured in these patients when there was no evidence of an acute viral infection or of malignant or any other disease associated with increased synthetase activity. In our Jan. 9 paper we suggested that there may be some defect in the IFN system in these patients. The data presented here do not necessarily rule this out. The similarity to the response in acute viral infections supports the existence of continuing viral infection in this group of patients.

INFLUENCE OF SOCIOECONOMIC STATUS ON BEHAVIORAL, EMOTIONAL AND COGNITIVE EFFECTS OF RUBELLA VACCINATION: A PROSPECTIVE, DOUBLE BLIND STUDY

A double-blind prospective design was used to investigate the immediate and prolonged psychological effects of a specific viral infection, and the role of immune activation in mediating these effects. Subjects were 240 female teenager girls who were vaccinated with rubella vaccine. Based on analysis of levels of antibodies to rubella, subjects were divided into two groups. An experimental group (n = 60), which included subjects who were initially seronegative and were infected following vaccination, and a control group (n = 180), which included subjects who were already immune to rubella before vaccination. Compared with the control group and to their own baseline, low socioeconomic status (SES) subjects within the experimental group showed a significant increase in the severity of depressed mood, social and attention problems, and delinquent behavior. Ten weeks post-vaccination there were no differences between the experimental and control groups in serum levels of interleukin-1 beta, interferon-gamma, soluble interleukin-2 receptors (sIL-2r), and cortisol. However, a significant negative correlation was found between fatigue-related symptoms and sIL-2r levels in the experimental (r = -0.325), but not the control group (r = -0.046). These findings suggest that viral infection can produce prolonged behavioral, emotional and cognitive problems mainly in subjects belonging to the low SES.

Immunoperoxidase method of identification ofLeishmania in routinely prepared histological sections

An indirect immunoperoxidase technique was applied for identification ofleishmania in routinely prepared histological sections. Paraffin embedded, formalin-fixed slide preparations of skin (1 case), bone marrow (2 cases) and lymph node (1 case) were examined. The tissues were obtained from one patient with cutaneous leishmaniasis and from three patients with visceral leishmaniasis. Specific rabbit anti-sera againstL. donovani, L. tropica andL. mexicana were used as primary reagents. Positive controls were performed simultaneously and includedL. tropica cultures in blood-agar (NNN media) - free promastigotes and amastigotes within macrophages. Strongly positive brown staining was localized specifically in Leishman-Donovan (LD) bodies only. This method increases the probability of microscopic diagnosis of leishmaniasis and helps to prevent confusion ofLeishmania with other infective agents in histological sections.

A sero-epidemiological study of herpes virus type 1 and 2 infection in Israel

In order to obtain data on the prevalence and incidence of herpes virus type 2 (HSV(2)) infection in selected populations of women and to identify groups that might benefit from routine prenatal screening, an epidemiological study was conducted during the period 1984-1990, which showed HSV(2) seroprevalence to be 2.8%. Due to the worldwide increase of over 30% of HSV(2) infection in the past two decades, a second study was performed during the period 1 January 1998-31 December 1999. Four different population groups were studied: 172 children aged 6 months to 17 years (group 1), 716 adults, men and women aged 18-95 (group 2), 200 women aged 30-67 who participated in the first survey and were re-examined in 1999 in the second survey (group 3), and a prevalence group of 155 parturient women from six different delivery rooms (group 4). Among the healthy 716 males and females HSV(2) seroprevalence was 4.5%. When analyzed by subgroup, HSV(2) seroprevalence rose from 2.3% in the 18-30 years subgroup to 6.5% in the 30-50 years subgroup and to 7.3% in the 51-70 years subgroup, and then declined to 2.4% after age 70 years. In the 200 women re-examined, HSV(2) seroprevalence was 7.7% with a 0.55% HSV(2) sero incidence per annum. In the prevalence group HSV(2) seroprevalence was 4.5%. Sera from the 1223 participants of all four groups were also screened for HSV(1) infection. HSV(1) antibody was present in 22% of children aged 6 months-1 year, in 60% at 21 years and in 87% at age 70 years. The data support the conclusion that in Israel there is no justification for routine prenatal HSV(2) screening in the healthy female population.

Extended nucleocapsid protein is cleaved from the Gag-Pol precursor of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 Gag and Gag-Pol precursors are translated from an mRNA which is indistinguishable from the full-length genomic RNA. The ratio of Gag to Gag-Pol polyproteins is approximately 20:1 and is controlled by a frameshift of the reading frame, which takes place downstream of the p7 nucleocapsid (NC) in the N terminus of the p1 peptide. The viral precursors Gag and Gag-Pol are cleaved by the virus-encoded protease (PR) into the structural proteins, and into p6(Pol), PR, reverse transcriptase and integrase. Due to the frameshift event, the cleavage site at the C terminus of NC coded in the Gag frame (ERQAN-FLGKI) changes either to ERQANFLRED or ERQANFFRED. The results presented in this report demonstrate that the NC released from the Gag-Pol precursor is 8 amino acid residues longer than the NC cleaved from the Gag polyprotein. Our results also show that truncated Gag-Pol precursors bearing cleavage site mutation at the NC/p6(Pol), and/or p6(Pol)/PR junctions, undergo autoprocessing in bacterial and eukaryotic cells, indicating that PR is active when part of the precursor.

Herpes simplex virus-1 and varicella virus infections in familial dysautonomia patients.

Familial dystautonomia (FD) patients are deficient in type C fibers, suggesting that there may be a different pattern of infection and clinical presentation when infected by Herpes simplex virus type 1 (HSV‐1) or Varicella‐Zoster virus (VZV). These viruses infect and are reactivated in the periphery of the body through type C sensory nerve fibers. HSV‐1 infects epithelial cells, penetrates into type C fibers, and migrates to the ganglia to generate latent infection. In reactivation, the viral DNA migrates through type C fibers, infecting the epidermis at the entry site. VZV infects through the respiratory tract, causing systemic viral infection and latency in the ganglia, from which it is reactivated and reaches the skin. The study was carried by clinical questionnaire and by HSV and VZV IgG antibodies on fifty‐one FD patients and eighty matched controls. The questionnaire revealed that no FD patient had a history of clinical HSV‐1 infection, compared to 15% in the control group (P < 0.05), while 50% FD patients had been infected by varicella, compared to 66% in the VZV control group. However in FD, VZV clinical manifestations were mild in comparison to controls. There was no difference in infection rates for some other viral diseases. HSV‐1 antibodies were detected in 24% of the FD patients, compared to 38% in the control group (P < 0.1). VZV antibodies were similar in FD and controls (66%, 63%). We concluded that the rate of HSV infection in FD is low and clinical reactivation is rare. The rate of varicella infection appears to be the same for patients and controls, but in FD the clinical presentation is mild. We suggest that these differences are due to the lack of type C fibers in FD patients. J. Med. Virol. 54:158–161, 1998.

Rapid isolation and identification of reovirus-3

Mammalian reoviruses are connected with a variety of humans diseases, including gastroenteritis, malabsorption and hepatitis. Recently, reovirus-3 was found to be associated with neonatal biliary atresia. We describe a technique for the rapid isolation and identification of reovirus-3. Mouse fibroblasts (L 929 cells) were grown in monolayers in a RPMI 1640 medium containing 10% calf serum. The cytopathic effects were visualized by the rounding of the L 929 cells and the appearance of fine granulation in the cytoplasm 48 h after the infection. Hematoxylin-eosin staining showed swelling and rounding of the infected cells, diminished chromatin in the nuclei, and the absence of mitoses. The immunohistochemical staining by the avidin-biotin-peroxidase technique was positive in the infected monolayers of the L 929 cells. The positive staining was limited to cytoplasmic inclusions, which were surrounded by a halo and sometimes by vacuoles. We conclude that the described technique is useful for the rapid isolation and identification of reovirus-3.