Innate immune responses to LCMV infections: natural killer cells and cytokines.

Although much remains to be learned, the study of early responses to LCMV infections of mice has contributed to the basic understanding of the regulation of a variety of important innate immune responses. Major discoveries have included the appreciation of the levels of type 1 IFNs induced during endogenous responses to viral infections, the importance of IFN-alpha/beta for induction of NK cell cytotoxicity, and the roles for IFN-alpha/beta in regulating the expression of other innate cytokines, i.e., IL-12 and IFN-gamma produced by NK cells (Fig. 11). Taken together with the characterization of adaptive responses to LCMV, a paradigm is emerging for a possible initial to innate to adaptive response cascade during infections with viruses eliciting endogenous expression of high levels of IFN-alpha/beta. The results not only advance the understanding of endogenous responses to viral infections and how they are balanced to achieve the best possible outcome for the host, but also give insights into possible consequences of therapeutic intervention with type 1 IFNs.

Increased apoptosis of T lymphocytes and macrophages in the central and peripheral nervous systems of Lewis rats with experimental autoimmune encephalomyelitis treated with dexamethasone.

Using light and electron microscopic histological and immunocytochemical techniques, we investigated the effects of the glucocorticoid dexamethasone on T cell and macrophage apoptosis in the central nervous system (CNS) and peripheral nervous system (PNS) of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) induced with myelin basic protein (MBP). A single subcutaneous injection of dexamethasone markedly augmented T cell and macrophage apoptosis in the CNS and PNS and microglial apoptosis in the CNS within 6 hours (h). Pre-embedding immunolabeling revealed that dexamethasone increased the number of apoptotic CD5+ cells (T cells or activated B cells), alphabeta T cells, and CD11b+ cells (macrophages/microglia) in the meninges, perivascular spaces, and CNS parenchyma. The induction of increased apoptosis was dose-dependent. Daily dexamethasone treatment suppressed the neurological signs of EAE. However, the daily injection of a dose of dexamethasone (0.25 mg/kg), which, after a single dose, did not induce increased apoptosis in the CNS or PNS, was as effective in inhibiting the neurological signs of EAE as the high dose (4 mg/kg), which induced a marked increase in apoptosis. This indicates that the beneficial clinical effect of glucocorticoid therapy in EAE does not depend on the induction of increased apoptosis. The daily administration of dexamethasone for 5 days induced a relapse that commenced 5 days after cessation of treatment, with the severity of the relapse tending to increase with dexamethasone dosage.

Effect of dosage regimen on natriuretic response to furosemide.

We have examined the differences in urinary excretion of water, sodium, potassium, chloride, urea, and creatinine produced by different dosage regimens offurosemide in normal volunteers. Three oral dosage regimens were compared: 20 mg daily, 20 mg twice daily, and 40 mg daily. Furosemide, 20 mg, did not produce a significant weight loss, diuresis, or natriuresis in 12 normal subjects. With 40 there was a significant weight loss, diuresis, natriuresis, and chloruresis over 24 hr. Comparison of the divided regimen with 40 mg daily revealed significantly greater sodium excretion, and chloride excretion with 20 mg twice daily. The divided dosage regimen produced a different pattern of diuresis with most of the sodium and water excretion occurring after the second dose. These differences in response to different regimens are predictable from pharmacokinetic considerations and may have clinical significance.

Construction and characterization of a recombinant adenoviral vector expressing human interleukin-12.

Interleukin-12 (IL-12) is a 70-kDa heterodimeric cytokine composed of a 35-kDa subunit (p35) and a 40-kDa subunit (p40). We have demonstrated previously that intratumoral delivery of a recombinant adenoviral vector expressing the mouse IL-12 gene significantly prolongs the survival time of mice with metastatic colon carcinoma in the liver. We now report the molecular cloning of cDNA for both subunits of human IL-12 (hIL-12) in a recombinant adenoviral vector in which the p40 and p35 subunits are linked and coexpressed using the encephalomyocarditis virus internal ribosome entry site. The recombinant adenoviral vector was used to transduce human tumor cell lines, and the presence of hIL-12 in the conditioned media was illustrated by enzyme-linked immunosorbent assay. The biological activity of hIL-12 in the conditioned media was also demonstrated in vitro through its ability to induce interferon-gamma production from peripheral blood mononuclear cells (PBMCs), to stimulate PBMC proliferation, and to enhance natural killer activity from normal human PBMCs to lyse natural killer-sensitive K562 target cells. The results of these studies support the application of this recombinant adenoviral vector construct as an efficient gene delivery vehicle in phase I/II clinical studies of hIL-12 gene therapy for cancer.

Demyelination and early remyelination in experimental allergic encephalomyelitis passively transferred with myelin basic protein-sensitized lymphocytes in the Lewis rat.

Histological studies were performed on Lewis rats with experimental allergic encephalomyelitis (EAE) passively transferred by myelin basic protein (MBP)-sensitized syngeneic spleen cells in order to determine the relationship between demyelination and neurological signs. Neither inflammation nor demyelination was present on the day prior to the onset of neurological signs but both were present in the spinal roots and spinal cord on the day of onset of tail weakness (4 days after passive transfer). Demyelination and the neurological signs both increased over the next 48 h. There was evidence that the caudal roots were more severely affected than the rostral roots. The peripheral nerves were spared. Demyelination in the spinal cord was concentrated in the dorsal root entry and ventral root exit zones. The initial stages of repair of demyelinated spinal root fibres by Schwann cells were observed on the earliest day that clinical recovery commenced (day 7). At this time some demyelinated fibres were closely associated with debris-free Schwann cells, and occasional fibres were completely invested by 1-2 layers of Schwann cell cytoplasm. Remyelination (compact myelin lamellae formation) by Schwann cells was first observed in the spinal roots on day 9. By the time of complete clinical recovery (day 11) the majority of affected spinal root cores had thin new myelin sheaths. Repair of central nervous system myelin by oligodendrocytes was slower than peripheral nervous system myelin repair. Investment of demyelinated spinal cord axons by oligodendrocytes was observed on day 9, and remyelination by these cells was seen on day 10. We conclude that the neurological signs of passively induced MBP-EAE can be accounted for by demyelination of the lumbar, sacral and coccygeal spinal roots and spinal cord root entry and exit zones, and that the subsequent clinical recovery can be explained by investment and remyelination of demyelinated peripheral and central nervous system fibres by Schwann cells and oligodendrocytes respectively.

Rapid entry and downregulation of T cells in the central nervous system during the reinduction of experimental autoimmune encephalomyelitis.

We investigated the mechanisms whereby a previous attack of experimental autoimmune encephalomyelitis (EAE) modifies a subsequent attack in the Lewis rat. Active immunization with myelin basic protein (MBP) and complete Freund's adjuvant 28 days after the passive transfer of MBP-sensitized spleen cells induced a second episode of EAE, which occurred earlier than in naive control animals, but was less severe overall. The pattern of neurological signs was also different in rechallenged rats, which had less severe tail and hindlimb weakness but more severe forelimb weakness. In rechallenged rats, inflammation was more severe in the cervical spinal cord, cerebellum, brainstem and cerebrum, but less severe in the lumbar spinal cord, than in controls. The early onset of EAE in rechallenged rats was explained by a memory T cell response to MBP(72-89) in the draining lymph node and spleen, and by the enhanced entry of T cells into the central nervous system (CNS). However, the number of alphabeta T cells in the spinal cord of rechallenged rats declined faster than in controls, especially in the lumbosacral cord, where the number of Vbeta8.2(+) T cells and the frequency of T cells reactive to MBP(72-89) rapidly decreased, indicating rapid downregulation of the immune response in the previously inflamed spinal cord. Apoptosis of inflammatory cells in the CNS was increased in the rechallenged rats and is likely to contribute to this downregulation. Furthermore, during the disease course the generation of encephalitogenic T cells in the peripheral lymphoid organs was limited compared with controls. Thus, a previous attack of EAE modifies a subsequent attack through the interaction of the following processes: a memory T cell response to MBP; facilitated T cell entry into the CNS; downregulation of the immune response in the CNS, including increased apoptosis of inflammatory cells; and a limited generation of encephalitogenic T cells in the peripheral lymphoid organs.

A simple technique for flat osmicating and flat embedding of immunolabelled vibratome sections of the rat spinal cord for light and electron microscopy.

We describe here a simple technique for flat-osmicating and flat-embedding of immunolabelled vibratome sections. The technique is particularly useful for large specimens such as whole cross-sections of rat spinal cord. After the vibratome section has been flat-osmicated on a flat surface under a glass coverslip, it is dehydrated and then embedded by placing it in a small drop of epoxy resin on a flat base of polypropylene plastic and overlaying a small square piece of cellulose acetate cut from heat-resistant overhead projector transparency film for photocopying. The thin layer of resin containing the flat-embedded vibratome section is then separated from the base and glued onto the flat end of a pre-polymerised blank block of resin. The method produces flat-embedded vibratome sections and thus allows serial large uniformly labelled semithin and ultrathin sections to be obtained of the whole cross-section of the rat spinal cord. This facilitates the observation and quantification of labelled cells in the specimen. Because of its simplicity the technique also allows one worker to process more than 100 vibratome sections at the one time.

Apoptosis in the nervous system in experimental allergic encephalomyelitis.

We report here for the first time the occurrence of apoptosis of cells in the spinal cord in experimental allergic encephalomyelitis (EAE), an autoimmune, T-cell-mediated demyelinating disease. Four different forms of EAE were studied in the Lewis rat: (i) acute EAE induced by inoculation with whole spinal cord and adjuvants; (ii) acute EAE induced by inoculation with myelin basic protein (MBP) and adjuvants; (iii) acute EAE induced by the passive transfer of MBP-sensitized spleen cells; (iv) chronic relapsing EAE induced by inoculation with whole spinal cord and adjuvants followed by treatment with low-dose cyclosporin A. Cells undergoing apoptosis were recognized at light and electron microscopy by the presence of either crescentic masses of condensed chromatin lying against the nuclear envelope or rounded masses of uniformly dense chromatin. They were found in both the white and grey matter of the spinal cord in all 4 forms of this disease. Although it was not possible to identify definitively the types of cells undergoing apoptosis, the size and location of some of the affected cells suggested that they were oligodendrocytes. As there is now a large body of evidence that T-cell-induced target cell death takes the form of apoptosis, it is attractive to hypothesize that oligodendrocyte apoptosis is occurring in EAE as a result of oligodendrocyte-directed T-cell cytotoxicity. However, other apoptotic cells were located within the myelin sheath, meninges and perivascular spaces and were clearly not oligodendrocytes but were most likely blood-derived mononuclear cells. The sparsity of their cytoplasm and the absence of phagocytosed material suggested that they were mainly lymphocytes rather than macrophages. Apoptosis has been shown to be involved in deleting autoreactive T-cells during the normal development of tolerance. Thus apoptotic deletion of myelin/oligodendrocyte-specific lymphocytes in the central nervous system in EAE might explain both the subsidence of inflammation and the acquisition of tolerance in this autoimmune disease.

Pathogenicity of Steinernema scapterisci to Selected Invertebrates.

Steinernema scapterisci was more pathogenic to insects tested in the order Orthoptera than to those in the orders Lepidoptera or Hymenoptera; it was not pathogenic to earthworms. The nematode also infected and killed the mole crickets Scapteriscus acletus and S. vicinus when released four successive times at 10-day intervals in containers of soil infested with the nematode.

Steinernema neocurtillis n. sp. (Rhabditida: Steinernematiclae) and a Key to Species of the Genus Steinernema.

Steinernema neocurtillis n. sp. isolated from the mole cricket Neocurtilla hexadactyla Perty can be distinguished from other members of the genus by characteristics of the first-generation male and the third-stage infective juvenile (IJ). In the male, the distance from the anterior end to the excretory pore (DAE) is less than the body width at the excretory pore; D% (DAE divided by length of esophagus x 100) is low at 19. The gubernaculum legth is greater than three-fourths the spicule length. Range of the ratio gubernaculum length divided by spicule length is 0.82-0.93 in the first-generation male and 0.92-1.00 in the second-generation male. In the IJ, the distance from the anterior end to the excretory pore is extremely short (18 mum), causing the D% and E% (DAE divided by tail length x 100) to be low (D% = 23 and E% = 12). Average body length of the IJ is 885 mum.

Life Cycle of Steinernema scapterisci Nguyen &Smart, 1990.

The life cycle of Steinernema scapterisci Nguyen and Smart, 1990 consists of an egg stage, four juvenile stages, and an adult stage (male and female). The cycle from IJ (third stage infective juveniles) to IJ may proceed by one of two routes. If the nutrient supply is sufficient and the population is not overcrowded, the IJ develop to adult males and females of the first generation. Most eggs from these adult females hatch and the juveniles develop through each life stage to become adult males and females of the second generation. Eggs produced by these females develop to IJ. This cycle takes 8-10 days (long cycle) at 24 C. If the nutrient supply is insufficient or if overcrowded, the IJ develop to adult males and females of the first generation, and eggs produced by the females develop directly to IJ. This cycle takes 6-7 days (short cycle). The nematode is less tolerant of lower temperatures and more tolerant of higher temperatures than are other species of the genus. The sex ratio is influenced by temperature. At 15 and 24 C, females constituted 54% and 60% of the population, respectively, but at 30 C females constituted 47% of the population.

Steinernema scapterisci n. sp. (Rhabditida: Steinernematidae).

Steinernema scapterisci n. sp., isolated in Uruguay from the mole cricket Scapteriscus vicinus, can be distinguished from other members in the genus by the presence of prominent cheilorhabdions, an elliptically shaped structure associated with the excretory duct, and a double-flapped epitygma in the first-generation female. The spicules of the male are pointed, tapering smoothly to a small terminus, and the shaft (calomus) is long, bearing a sheath. The gubernaculum has a long, upward-bent anterior part. The ratio of head to excretory pore divided by tail length of the third-stage juvenile is greater for S. scapterisci n. sp. than for S. carpocapsae. Steinernema scapterisci n. sp. did not hybridize with S. carpocapsae strain Breton. In laboratory tests, S. scapterisci n. sp. killed 10% or less of non-orthopteran insects, including the wax moth larva, a universal host for other species of Steinernema.

Vertical Dispersal of Steinernema scapterisci.

When infective juveniles ofSteinernema scapterisci Nguyen &Smart were released on the soil surface in the field and in the laboratory, some of them moved downward through the soil at least 10 cm in 5 days and infected and killed mole crickets. When released 2 cm below the soil surface, most of the juveniles moved into the upper 2 cm layer of soil, but some moved downward 10 cm. When placed at the center of a 16-cm soil column, infective juveniles moved in both directions with three times more moving downward than upward. Infective juveniles were more efficient in killing mole crickets in the field than in the laboratory.

Identification of entomopathogenic nematodes in the steinernematidae and heterorhabditidae (nemata: rhabditida).

This paper contains taxonomic keys for the identification of species of the genera Steinernema and Heterorhabditis. Morphometrics of certain life stages are presented in data tables so that the morphometrics of species identified using the keys can be checked in the tables. Additionally, SEM photographs and diagnoses of the families and genera of Steinernematidae and Heterorhabditidae are presented.

Rhabditis (Oscheius) pheropsophi n. sp. (Rhabditida: Rhabditidae).

Rhabditis (Oscheius) pheropsophi n. sp., associated with cadavers of the bombardier beetle, Pheropsophus aequinoctialis, is described from material collected in Brazil. Mean body length of the female is 1,217 mum, of the male 872 mum, and of the dauer juvenile 568 mum. The female has six lips with one papilla on each lateral lip and two on each sublateral lip; stoma wall thickened dorsally, metarhabdions with warts, excretory pore near base of esophagus, tail long (c = 8), and phasmids prominent, protruding on scanning electron microscope preparations. The male has 10 pairs of bursal ribs, with the terminal pair considerably smaller than the others; spicules fused distally two-thirds of their length. The new species can be distinguished from other members of the Dolichura-group by its fused spicules.

Neosteinernema longicurvicauda n. gen., n. sp. (Rhabditida: Steinernematidae), a Parasite of the Termite Reticuldermes flavipes (Koller).

A nematode isolated from the termite Reticulitermes flavipes (Koller) was identified and described as a new genus and species, Neosteinernema longicurvicauda. Primary distinguishing characters, by contrast to members of the genus Steinernema, were females having prominent phasmids, a curved tail longer than the body width at the anus, a spiral shape in juvenile-bearing females, and juveniles becoming infective-stage juveniles before emerging from the female; males having prominent phasmids, a digitate tail tip, a characteristic shape of the spicules (foot-shaped with a hump on the dorsal side), and 13-14 pairs of genital papillae, with eight pairs preanal; and infective juveniles having prominent phasmids and a filiform curved tail as long as the esophagus. Adult nematodes are found outside the termite cadaver. Diagnosis of the family Steinernematidae was emended to accommodate the new species.

Paraiotonchium muscadomesticae n. sp. (Tylenchida: lotonchiidae) a Parasite of the House Fly (Musca domestica) in Brazil and a Key to Species of the Genus Paraiotonchium.

Paraiotonchium muscadomesticae n. sp., a parasite of the house fly, Musca domestica L., is described and illustrated from material collected in Brazil. The life cycle of P. muscadomesticae is similar to that of P. autumnale (Nickle), consisting of alternating gamogenetic and parthenogenetic generations. Paraiotonchium muscadomesticae n. sp. can be distinguished from P. nicholasi Slobodyanyuk, P. autumnale (Nickle) Slobodyanyuk, and P. crassirostris (Yatham &Rao) Siddiqi by the shorter body length of young heterosexual females, 652 g.m (530-709) for P. muscadomesticae compared to 750 mum or more (801-1,050) for the others. Paraiotonchium muscadomesticae is close to P. nicholosi but differs from it by V ratio and spicule length (V = 80-84; spicule = 16-21 p.m in P. muscadomesticae compared to V = 73-78; spicule = 25-35 mum in P. nicholasi). Paraiotonchium muscadomesticae and P. nicholasi differ from all species of this genus by the absence of a bursa on males of these two species.

Scanning Electron Microscope Studies of Steinernema anomali Kozodoi, 1984.

This paper presents SEM micrographs of portions of the male, female, and infective-stage juvenile of Steinernema anomali. Included are micrographs of the cephalic and caudal region, spicules, and gubernaculum of the male, the cephalic and vulval region of the female, and the cephalic region of the infective-stage juvenile. Males have six labial and four prominent cephalic papillae and small amphids. There are 11-14 pairs and one single genital papillae; of these, 6-9 pairs are preanal and subventral, one pair preanal, lateral, one pair adanal, and three pairs postanal. Spicules have a short head, a long blade, and a reduced shaft. The distal end is enlarged and bears a dorsal aperture. Gubernaculum much shorter than spicules; cuneus of gubernaculum short and bifurcate anteriorly. Females have six labial and four cephalic papillae and small amphids. Vulva with a thickened posterior lip. Infective juveniles have a smooth head, prominent amphids, and four cephalic papillae. Labial papillae, if present, are not evident.

Location of the Phasmids on Infective Juveniles of Steinernema glaseri.

Scanning electron microscopy revealed the location of the phasmids on infective juveniles of Steinernema glaseri. The phasmids are located about 40% of the tail length posterior to the anus and are at or near the same level. Instead of being in the center of the lateral fields, they are located just ventral to the lateral fields, or interrupting the ventral-most lateral ridge. The phasmids were covered often by an exudate.

Morphometrics of Infective Juveniles of Steinernema spp. and Heterorhabditis bacteriophora (Nemata: Rhabditida).

Selected morphometrics of Heterorhabditis bacteriophora and seven species of Steinernema from in vivo culture were compared in relation to time of harvest. In addition, five Steinernema species were reared in vitro and their morphometrics were compared with those from in vivo culture. With in vivo culture, there was generally a negative linear relationship between body length of infective juveniles (IJ) and time of harvest. The distance from the anterior end to the excretory pore (EP) and the tail length (T) of IJ also varied with time of harvest. The E percentage (= EP/T x 100) was the least variable. Body lengths of IJ reared in vitro were much less than those of IJ reared in vivo. The study suggests that IJ harvested from in vivo culture within 1 week of emergence from cadavers are best for species identification. Infective juveniles from in vitro culture should not be used for species identification.