EXPERIMENTAL TYPE III PNEUMOCOCCUS PNEUMONIA IN MONKEYS : II. TREATMENT WITH AN ENZYME WHICH DECOMPOSES THE SPECIFIC CAPSULAR POLYSACCHARIDE OF PNEUMOCOCCUS TYPE III

The effects of specific enzyme therapy upon experimental Type III pneumococcus pneumonia in monkeys were studied by comparing the course and outcome of the disease in treated animals with that in animals which received no therapeutic aid. Enzyme treatment was found to exert a distinctly favorable influence upon the experimental pneumonia. Treatment was followed by cessation of spread of the pneumonic lesion, sterilization of the blood, and early recovery, except in animals in which the severity of the disease was extreme. While in the untreated animals a high incidence of empyema and pericarditis was observed, suppurative sequelae were apparently prevented by adequate enzyme therapy. The limitations of the therapeutic action of the specific enzyme in the presence of marked depression of the cellular reaction in infected animals are again emphasized.     

PROPERTIES OF THE CAUSATIVE AGENT OF A CHICKEN TUMOR : IV. ASSOCIATION OF AN INHIBITOR WITH THE ACTIVE PRINCIPLE

The presence of an inhibiting substance in the chicken tumor is shown by the fact that a desiccate of the tumor is more active after it has been washed two or three times with water, and that an extract of the tumor is more potent after some factor is removed by adsorption on aluminum hydroxide. When the tumor-producing factor in an extract of a slow-growing tumor has been destroyed by heating at 55°C. it is found to have the property of neutralizing a highly active tumor extract. This inhibiting property is destroyed by heating over 65°C.     

CHANGING VIRAL SUSCEPTIBILITY OF A HUMAN CELL LINE IN CONTINUOUS CULTIVATION : I. PRODUCTION OF INFECTIVE VIRUS IN A VARIANT OF THE CHANG CONJUNCTIVAL CELL FOLLOWING INFECTION WITH SWINE OR N-WS INFLUENZA VIRUSES

During its serial transfer and cultivation in this laboratory, a human conjunctival cell line (Chang) was observed to change in morphology. Concurrently no change was noted in the susceptibility of the cells to viruses capable of infecting the original cell line. However, it was noted that the derived variant cell line had acquired susceptibility to the induction of cytopathic effects and incomplete virus formation by several strains of influenza viruses. It was then discovered that swine influenza virus and the N-WS strain of influenza A virus could be serially propagated in the derived cell line with production of infective virus. The swine virus required adaptation, but the N-WS strain did not. N-WS and swine influenza viruses multiply with infective virus formation only in the variant conjunctival cell and in no other cell line. Antigenic, cytologic, and virologic evidence is presented that the influenza virus-susceptible variant cell is of human origin and is not a contaminating cell exogenously introduced. Transition of a cell line from complete insusceptibility to susceptibility to virus infection and multiplication has not been described previously.     

Leukocyte Glucocerebrosidase Deficiency Diagnostic in Adult Gaucher's Disease with Negative Bone Marrow Biopsy. Some Properties of the Enzyme in Leukocytes and Spleen

Abstract. A sixty-one year-old man with Gaucher's disease is described. The disease was manifested by splenomegaly, hypersplenism and elevations of serum acid phosphatase and immunoglobulin but no bone lesions were roentgenologically demonstrable and bone marrow biopsy failed to reveal Gaucher cells. The diagnosis was established prior to splenectomy by the finding of decreased β-D-gIucoside glucohydrolase (β-D-GGH) activity in his peripheral leukocytes. Examination of the surgically removed spleen revealed Gaucher cells, a large excess of glucocerebroside and decreased β-D-GGH activity. Splenectomy was followed by normalization of haemoglobin, white cell and platelet counts and of the serum acid phosphatase and immunoglobulin levels. The patient's leukocyte β-D-GGH activity showed an optimum pH similar to that of normal leukocyte enzyme, about 5.4, using both glucocerebroside (Glc-Cer) and 4-methylumbelliferyl-β-D-glu-copyranoside (MU-Glc) as substrate. The patient's spleen β-D-GGH activity showed a pH optimum of about 4.5, which differed slightly from that of normal spleen enzyme. The Km values of β-D-GGH in the patient's leukocytes for Glc-Cer and MU-Glc were 1.35 × 10^-4 M and 3.6 × 10^-3 M, respectively, i.e. in the normal range. The patient's splenic tissue Km values were for Glc-Cer and MU-Glc 0.52×10^-4M and 5.8×10^-3 M, respectively, as compared to 0.30×10^-4 M and 5.0 × 10^-3 M in a normal control.     

Role of Antibody and Complement in the Immune Clearance and Destruction of Erythrocytes II. MOLECULAR NATURE OF IgG AND IgM COMPLEMENT-FIXING SITES AND EFFECTS OF THEIR INTERACTION WITH SERUM

A model for the immune clearance and destruction of homologous erythrocytes has been further explored. In this model, every IgM anti-erythrocyte antibody molecule in an antibody preparation was shown to fix Cl. About 2000 IgG antibody molecules were required to form a Cl-fixing site on the guinea pig erythrocyte surface. 60 IgM complement-fixing sites per erythrocyte were required for the immune clearance of IgM-sensitized erythrocytes. This number of sites could be detected by a direct agglutination test. 1.4 complement-fixing sites were required for immune clearance of IgG-sensitized cells, a number of molecules which could not be detected by direct agglutination. This number could, however, be detected with the use of a Coombs antiglobulin reagent.Depletion of the late components of complement (C3-9) with cobra venom was associated with the loss of ability to clear IgM-sensitized cells and a marked deficit in the ability to clear IgG-coated cells. Thus, late (C3-9) components of complement as well as an early component (C4) were required for normal clearance of sensitized erythrocytes. There was no evidence that activation of the alternate pathway of complement action could lead to accelerated erythrocyte clearance.In vitro incubation of IgG and IgM-sensitized erythrocytes in fresh serum led to deposition of C3 and C4 on the erythrocyte surface. IgM-sensitized cells treated in this way had a normal survival. IgM-sensitized cells also were shown to remain Coombs positive after their release from the liver. The evidence suggests that the interaction of an IgM site with fresh serum in vitro and in vivo leads to formation of a site which allows for sequestration of cells in the liver. With continued exposure to serum components, this site is destroyed or inactivated. This serum-dependent inactivation is complement-dependent as shown by the use of EDTA-treated and C4-deficient serum. IgG complement-fixing sites are only partially inactivated by incubation in fresh serum, further emphasizing the differences in the biologic activity of IgM and IgG antibodies.     

AN IMMUNIZING SUBSTANCE FOR SEMLIKI FOREST VIRUS IN THE LIVERS OF IMMUNE MICE : I. THE INITIAL OBSERVATION TOGETHER WITH A CONSIDERATION OF THE ATTENDING CONDITIONS

A material capable of rendering mice resistant to Semliki Forest virus is present in the livers but not in the muscles, brains, or spleens of mice that have survived a usually fatal dose of virus.     

Serum Thyrotropin Responses to Synthetic Thyrotropin-Releasing Hormone in Normal Children and Hypopituitary Patients. A NEW TEST TO DISTINGUISH PRIMARY RELEASING HORMONE DEFICIENCY FROM PRIMARY PITUITARY HORMONE DEFICIENCY

Synthetic thyrotropin-releasing hormone (TRH) was administered intravenously in a dose of 7 mug/kg to 20 normal children ages 4-13 yr. Serum thyroid-stimulating hormone (TSH) was measured by radioimmunoassay and rose from a mean value of 1.7 muU/ml (range = < 1.25-7.2) to a mean peak value of 21.5 muU/ml (5.2-33.2) at 15 or 30 min after administration.13 patients with idiopathic hypopituitarism and apparent normal thyroid function, ages 3-19 yr, responded to TRH in a manner very similar to the control subjects: TSH rose from a mean value of 1.8 muU/ml (range < 1.25-4.3) to a mean peak value of 18.5 muU/ml (range = 9.5-45.0) which occurred between 15 and 60 min after TRH.13 idiopathic hypopituitary patients with documented thyroid deficiency were tested after thyroid therapy had been discontinued for a minimum of 10 days. The serum TSH values in 10 of 13 patients rose from a mean base line level of 2.2 muU/ml (< 1.25-5.3) to a peak mean value of 32.5 muU/ml (9.6-61.3) between 30 and 120 min after TRH. In three patients, however, little or no TSH response was detected, even when serum thyroxine levels were extremely low. Similar to the latter group, three of five patients with hypopituitarism secondary to craniopharyngiomas had undetectable or barely measurable TSH levels before and after TRH. Two of these five patients had significant responses which were compatible with hypopituitarism resulting from damage to the hypothalamus or hypothalamic vessels instead of the pituitary.Side effects were experienced in 41 of 54 patients (76%). The effects were limited to a mild nausea-like sensation in 63% of the patients and occurred within the first 5 min after receiving TRH. No evidence of serious toxicity or long-term side effects was noted.The TRH test is a safe, effective way to measure TSH reserve in children. The positive response in 10 of 13 patients with secondary hypothyroidism supports data previously accumulated that most patients with idiopathic hypopituitarism have an abnormality of their hypothalamic-releasing hormone function, whereas the remaining minority probably have primary pituitary disease.     

STUDIES ON THE IMMUNE RESPONSE OF THE RHEUMATIC SUBJECT AND ITS RELATIONSHIP TO ACTIVITY OF THE RHEUMATIC PROCESS : III. OBSERVATIONS ON THE REACTIONS OF A RHEUMATIC GROUP TO AN EPIDEMIC INFECTION WITH HEMOLYTIC STREPTOCOCCUS OF A SINGLE TYPE

This study of an isolated colony showed that of seven children who escaped the epidemic streptococcus infection none developed rheumatic symptoms; and that of seventeen children who contracted the epidemic streptococcus infection, fourteen developed acute rheumatism and three showed no recognizable rheumatic manifestations. The seven children who failed to contract infection with Streptococcus hemolyticus showed clearly that susceptible individuals may live in dose association with an epidemic of acute rheumatism, develop no rise in antistreptolysin titer and maintain excellent health. The patient with congenital heart disease demonstrated that a non-rheumatic subject may be infected with a highly effective strain of hemolytic streptococcus, and develop a typical antibody response, yet escape all rheumatic manifestations. The two patients who, although infected with the epidemic strain, failed to show any antibody response, also failed to develop rheumatic recrudescences. Environmental, dietary, age and the other factors investigated did not appear to be significant in this outbreak of acute rheumatism. Three factors appeared to determine the development of the fourteen recrudescences: (1) infection with a highly effective agent; (2) the disease pattern, peculiar to each rheumatic subject; (3) the intensity of the immune response of the patient as indicated by the rise in antistreptolysin titer.     

Role of Antibody and Complement in the Immune Clearance and Destruction of Erythrocytes I. IN VIVO EFFECTS OF IgG AND IgM COMPLEMENT-FIXING SITES

A model which permits evaluation in molecular terms of the role of antibody and of complement in the immune destruction of erythrocytes was established in the guinea pig. IgM and IgG immunoglobulins were isolated from rabbit anti-guinea pig erythrocyte antisera and were used to sensitize ⁵¹Cr-labeled guinea pig erythrocytes. The average number of complement-fixing sites per erythrocyte formed by antibody was determined for each of the various preparations by the Cla fixation and transfer test. The rate of clearance and of organ localization was determined for cells sensitized with either IgM or IgG antibodies, and dose-response curves were established in normal guinea pigs and guinea pigs with a genetically controlled, complete absence of the fourth component of complement (C4).     

Activity of selenazofurin against influenza A and B viruses in vitro.

Activity of the new antiviral compound selenazofurin was compared with the known active compounds ribavirin and amantadine against influenza A and B viruses. In experiments with Madin Darby canine kidney cells, selenazofurin inhibited the cytopathic effect and yield of influenza A/NWS/33 virus, with 50% effective dose ranges of 0.7 to 1.4 micrograms/ml (virus rating [VR], 1.3 to 1.4). The 50% effective dose range for ribavirin was 1.2 to 1.6 micrograms/ml (VR, 1.0 to 1.3), and for amantadine it was 9 micrograms/ml (VR, 0.9). Selenazofurin and ribavirin were similarly inhibitory to influenza B/Lee/40 virus, whereas amantadine was inactive. Selenazofurin appeared somewhat cytotoxic in these studies at concentrations as low as 1 micrograms/ml.     

Assessment of Pandemic and Seasonal Influenza A (H1N1) Virus Susceptibility to Neuraminidase Inhibitors in Three Enzyme Activity Inhibition Assays

The neuraminidase inhibitors (NAIs) zanamivir and oseltamivir are currently the only antiviral drugs effective for the treatment and prophylaxis of 2009 pandemic influenza A (H1N1) virus infections. The proven potential of these viruses to acquire NAI resistance during treatment emphasizes the need to assess their NAI susceptibility. The 50% inhibitory concentrations (IC₅₀s) are known to vary depending on the neuraminidase inhibition (NI) test used; however, few side-by-side comparisons of different NI assays have been done. In the present study, a panel of 11 isolates representing 2009 seasonal and pandemic influenza H1N1 viruses, including oseltamivir-resistant H275Y variants, were tested in three functional NI assays: chemiluminescent (CL), fluorescent (FL), and colorimetric (CM). The sensitivities of the viruses to zanamivir, oseltamivir, and three investigational NAIs (peramivir, R-125489, and A-315675) were assessed. All isolates with the exception of H275Y variants were sensitive to all five NAIs by all three NI assays. The H275Y variants showed substantially elevated IC₅₀s against oseltamivir and peramivir. The three NI assays generally yielded consistent results; thus, the choice of NI assay does not appear to affect conclusions based on drug susceptibility surveillance. Each assay, however, offers certain advantages compared to the others: the CL assay required less virus volume and the FL assay provided the greatest difference in the IC₅₀s between the wild type and the variants, whereas the IC₅₀s obtained from the CM assay may be the most predictive of the drug concentrations needed to inhibit enzyme activity in humans. It would be desirable to develop an NI assay which combines the advantages of all three currently available assays but which lacks their shortcomings.For the treatment and chemoprophylaxis of infections caused by influenza A viruses, the U.S. Food and Drug Administration (FDA) has approved four drugs: amantadine and rimantadine as well as zanamivir and oseltamivir. These drugs belong to two classes, adamantanes (i.e., M2 ion-channel blockers) and neuraminidase (NA) inhibitors (NAIs), respectively. In recent years, the effectiveness of M2 blockers has been greatly compromised, which limits their usefulness in clinical practice. This is largely due to the rapid emergence and widespread circulation of adamantane-resistant influenza viruses (1, 5, 6, 7, 14, 17). More recently, the emergence and worldwide spread of seasonal H1N1 viruses resistant to oseltamivir, currently the most widely used drug against influenza infections, became a considerable public health concern (15, 21, 25, 32). Monitoring the NAI resistance of influenza viruses is an ongoing public health issue since the emergence in 2009 of pandemic viruses that are resistant to M2 blockers.Cell culture-based assays are typically not used for assessment of virus sensitivity to NAIs because of the unpredictable effect of hemagglutinin (HA) receptor binding (2, 34). Instead, drug susceptibility can be monitored by functional (biochemical) NA inhibition (NI) assays, and subsequent genotypic methods are generally required to identify the molecular marker(s) of resistance in the NA. The principle underlying the functional methods relies on the enzymatic nature of the NA, a viral surface glycoprotein and antigen. NA acts by cleaving the terminal neuraminic acid (also called sialic acid) from receptors recognized by influenza viral HA, thus facilitating the release of progeny virions from infected cells and preventing self-aggregation (29). Structurally, NAIs mimic the natural substrate, neuraminic acid, and produce tight interactions, with conserved residues of the NA active site competing with neuraminic acid for binding (11, 23). Preincubation of virus with NAIs leads to the inhibition of enzyme activity, which is detected after the addition of enzyme substrate. Most NI assays commonly used for virus surveillance utilize as substrates small synthetic conjugates that produce either a luminescent or a fluorescent signal upon cleavage by the NA enzyme. The chemiluminescent (CL) assay uses the 1,2-dioxetane derivative of neuraminic acid substrate in the influenza neuraminidase inhibitor resistance detection (NA-Star) kit (8), while the fluorescent (FL) assay employs 2′-O-(4-methylumbelliferyl)-N-acetylneuraminic acid substrate (MUNANA) (30). The results of the NI assays are expressed as the 50% inhibitory concentration (IC₅₀), which represents the NAI concentration that inhibits 50% of the enzyme activity of the virus. As the NA activity of clinical specimens is usually insufficient for determining the IC₅₀ due to a low viral content, NI assays, using either the substrate provided with the NA-Star kit or the MUNANA substrate, require virus propagation in cell cultures or embryonated chicken eggs. It is noteworthy that IC₅₀s are specific to the virus type/subtype and to the individual NAI tested (8, 19, 20, 24, 32, 37). The IC₅₀s obtained can be used for assessment of virus susceptibility to NAIs, including detection of resistant viruses, as well as for comparing the potencies of antiviral drugs belonging to the NAI class. Although both the CL and FL assays allow reliable detection of NAI resistance, the more recently developed CL assay was reported to be about 70 times more sensitive in detecting NA activity and has a greater linear range than the FL assay (8). The CL assay was also selected for use in the global drug susceptibility surveillance program by the Neuraminidase Inhibitor Susceptibility Network (NISN) (37, 39) and by other surveillance laboratories (28, 32). It should also be noted that IC₅₀s may vary even for the same virus when the NI assay is done using the NA-Star substrate (CL assay) and the MUNANA substrate (FL assay), according to reports on seasonal viruses (37). Whether one of the two assays, the CL or FL assay, more reliably predicts the level of resistance and the drug concentration required for the NA activity inhibition in vivo are key points of interest and remain to be elucidated.A third assay, the colorimetric (CM) assay, which utilizes fetuin as the substrate of the NA, is typically used to determine the titer of anti-NA antibodies because small substrates do not effectively compete with antibodies (3, 31). This assay is not widely used for antiviral susceptibility testing. Unlike the NA-Star and MUNANA synthetic substrates, fetuin is a large, natural, and soluble bovine glycoprotein that contains abundant neuraminic acids at the ends of its oligosaccharide moiety (which include the presence of two residues of α2,3-linked sialic acid and one residue of α2,6-linked sialic acid) (4, 33) and has been used as a substrate in NA-catalyzed reactions (3). Given that NAIs compete with the enzyme substrate for binding to the active site, the structure of the substrate can potentially influence the outcome of the competition and, as a result, the IC₅₀. In this respect, fetuin may represent a better natural substrate for the enzyme-neuraminic acid attached via an α2,3 or α2,6 linkage to oligosaccharide chains on the cell surface. Furthermore, since the cleavage of each neuraminic acid is chemically converted, the CM assay can be a quantifiable method from which the resulting IC₅₀s would correlate more closely to the NA activity of the virus tested. Despite these apparent advantages to the use of fetuin, the CM method relies on chemical reactions that are time-consuming, cumbersome, and impractical for high-throughput use. In addition, the assay requires concentrated virus stocks for testing. Thus, fetuin is still considered an undefined substrate that does not confer sufficient sensitivity or specificity for use in routine NAI susceptibility assays (34). The potential usefulness of a large substrate such as fetuin for assessment of the NAI susceptibilities of novel H1N1 viruses or novel inhibitors remains largely unexplored.Resistance to NAIs is not defined as clearly as that to adamantanes. In NI assays, a drug-resistant virus should have IC₅₀s consistently greater than the threshold value that is determined for each viral type/subtype and drug tested (27, 32, 37). Since the 2007-2008 influenza season, about a decade after the introduction of NAIs into clinical use, an NA framework mutation, H275Y (H274Y in N2 numbering), was consistently and most commonly detected in oseltamivir-resistant H1N1 viruses isolated worldwide (15, 21, 25, 32). Although the H275Y substitution represents the most-defined oseltamivir resistance marker of influenza viruses carrying the NA of the N1 subtype (35), novel NAI resistance-associated mutations—determined by elevated IC₅₀s in NI assays—continue to be revealed (21, 22, 32). Importantly, oseltamivir-resistant viruses from the ongoing H1N1 pandemic have been detected and reported around the world (9, 10, 26, 38). Seasonal and 2009 pandemic H1N1 viruses have the same phylogenetically distant NA gene ancestors (16), which necessitates the comprehensive assessment of the drug susceptibilities of the new pandemic viruses. Therefore, it is necessary to evaluate existing NI assays in order to better understand which assay may be the most sensitive for the detection of NAI resistance and/or the most predictive of virus susceptibility to NAIs in vivo.In the present study, we assessed the susceptibilities of a panel of seasonal and pandemic H1N1 influenza viruses, including virus variants bearing the established oseltamivir resistance mutation, H275Y in the NA, against five NAIs: two FDA-approved NAIs, zanamivir and oseltamivir, and three investigational NAIs, peramivir, R-125489 (the bioactive metabolite of the prodrug CS-8958 [laninamivir]), and A-315675 (a bioactive form of the prodrug A-322278). In order to better characterize and assess the consistency of IC₅₀s and levels of susceptibility, these viruses were tested in the widely used CL and FL assays, as well as with the CM method.     

EFFECTS OF CONCANAVALIN A ON MOUSE PERITONEAL MACROPHAGES : I. Stimulation of Endocytic Activity and Inhibition of Phago-Lysosome Formation

Concanavalin A (Con A) binds to saccharide residues on the mouse peritoneal macrophage plasma membrane and stimulates extensive pinocytic interiorization of the membrane. The overall pinocytic rate is increased 3.5–4.5 times by the addition of Con A, and the surface marker enzyme adenosine triphosphatase can be identified histochemically in association with the cytoplasmic vesicles generated after exposure of the cells to Con A. Once formed, these pinocytic vesicles may persist for several days and fail to show morphologic evidence of fusion with primary or preformed secondary lysosomes. There is no apparent effect on the capacity of the macrophage to ingest either latex particles or IgG-coated SRBC administered either simultaneously with or subsequent to the Con A.