Transforming growth factor-β1 (TGFβ) is a peptide cytokine implicated in control of growth, adhesion, and differentiation of cells in numerous tissues. TGFβ has potent inhibitory effects on many lymphocyte responses; we also found that TGFβ could inhibit in vitro generation of tumor-specific CTL. Suppression was largely reversed with exogenous IL-2. Proliferation of memory-stage CTL clones was also inhibited by TGFβ, while the upregulation of cytotoxicity was not inhibited by TGFβ. These studies suggested that TGFβ limited anti-tumor CTL responses via both an indirect effect on IL-2 production, and by a direct effect on proliferation of memory CTL. In CTLL-2 cells, TGFβ inhibited IL-2-dependent DNA synthesis and cell growth as early as 24h after addition. TGFB inhibited IL-2-dependent surface IL-2Rα expression 24h after treatment, while cells remained 100% IL-2R+ up to 48h after treatment. TGFβ inhibited c-myc mRNA expression as early as 1h after treatment, suggesting that TGFβ may inhibit T cell growth by either altering signal transduction through the IL-2R, or by otherwise inhibiting early gene expression events triggered by IL-2 binding. TGFβ treatment also resulted in morphologic changes and increased adherence in 50% of CTLL-2 cells. Adherence required the presence of fetal calf serum and could be largely blocked by RGDS peptides. Under these conditions, de novo surface expression of CD8α and CD8β was observed and these cells rapidly accumulated mRNA encoding both CD8α and β chains, to a level 4-fold greater than control. Treatment of CD4-CD8- thymocytes with IL-2 + TGFβ similarly induced high levels of de novo CD8α expression on one-third of cells, while few thymocytes treated with IL-2 alone became CD8α+. CD8 expression in mature, peripheral CD8+ cells was not influenced by TGFβ. These data suggest that TGFβ has both positive and negative regulatory effects on the expression of molecules important for T lymphocyte growth, differentiation and function, and TGFβ may be a physiologically important cytokine for initial expression of CD8 in the thymus.

Primary amebic meningoencephalitis is a fatal disease of man caused by the free-living ameboflagelate Naegleria fowleri. In general, the victims have been active, healthy, young adults with a recent history of swimming or other fresh water-related activity. Infection with N. fowleri is apparently by way of nasal introduction of amebae-containing water. After nasal installation, electron microscopic and histopathologic studies with experimental animals reveal that amebae reside in the olfactory mucosa and then invade and migrate through the submucosal structures into the nerve plexuses. Amebae pass through pores of the cribiform plate and into the subarachnoid space. Subsequently, amebae invade the olfactory bulbs and lobes and spread to more distant areas of the brain causing massive hemorrhage, necrosis and edema. Frequently, the amebae aggregate in the perivascular spaces where they provoke a predominantly neutrophilic cellular response. Within seventy-two hours after the onset of symptoms, a rapid deterioration of the patient ensues resulting in coma and death (Carter, l972; Martinez et al., l977). Standard amebocides, such as diodohydroxyquin, chloroquin and metronidazole are ineffective in treating N. fowleri infections. Amphotericin B, an antibiotic used to treat systemic fungal infections, has been shown to be an effective antinaeglerial agent in vitro. Its ability to provide protection in vivo is unclear (Carter, l969; Padilla and Padilla, 1974; Schuster and Rechthand, l975). However, in Australia, Anderson and Jamieson (1972) used amphotericin B to successfully treat a l4 year old boy with confirmed primary amebic memingoencephalitis. Duma et al. (l97l), using the same drug and similar regimen, were unsuccessful with two patients at the Medical College of Virginia, Richmond. Other drugs, such as penicillin, suphadiazine, chloramphenicol, oxytetracycline HCl, streptomycin, methotrexate and emetine have had no effect on N. fowleri in vitro at levels in excess of those likely to be obtained therapeutically in the brain (Carter, 1968). The taxonomic position of Naegleria places it in the kingdom Protista, phylum Protozoa and class Sarcodina. It is of the order Schizopyrenida and the family Vahlkamphidae due to its ability to transform from trophozoite to flagellate and because of its promitotic nuclear division. Naegleria reproduction involves nuclear division (karyokinesis), in which the nucleolus elongates and divides into two polar masses and the nuclear membrane remains intact, followed by cytoplasmic division (cytokinesis). The genus Naegleria is identified by organisms which are biflagellate and do not possess cytochromes. Within the genus Naegleria there are two species, the pathogen N. fowleri and the nonpathogen N. gruberi (Page, l976). Willaert and Le Ray (l973) have described a third species, N. jadini. Synonyms for N. fowleri are N. aerobia (Singh and Das, l970) and N. invades (Chang, 197l). Synonyms for N. gruberi are Amoeba gruberi, Dimastingamoeba gruberi and N. punctata (Fulton, 1970). Naegleria fowleri can be differentiated from N. gruberi in many ways. The mitochondria of N. fowleri are dumbbell-shaped rather than oval as in N. gruberi. Naegleria gruberi cysts have numerous conspicuous pores through which excystment occurs while N. fowleri cysts have few inconspicuous pores. Naegleria fowleri cysts are less resistant to drying than are cysts of N. gruberi (Carter, 1970). Naegleria fowleri is pathogenic, grows best at 37 C, although it will grow at 45 C, and is unable to grow in the presence of 0.5% saline. Naegleria gruberi is nonpathogenic, grows best at 25 C and grows well with 0.5% saline in the medium (Singh and Das, 1970). Naegleria fowleri cysts are more sensitive to chlorine than are the cysts of y, gruberi DeJonckheere and Van de Voorde, 1976). These species a1so differ in optimal pH for growth, growth media composition and size of the amebae. Concanavalin A agglutinates fl, gruberi but not H, fowleri (Josephson et a1., 1977). Naegleria iggifli reportedly can be differentiated from E, fowleri by its reduced virulence and inability to grow at 37 C and from y, gruberi by its nonporus cyst wall ( Nillaert and LeRay, 1973). Researchers have used mice, guinea pigs, monkeys and rabbits in their investigations of experimental primary amebic meningoencephalitis. Probably the most useful laboratory animal model involves the mouse. Mice have been used because investigators have shown that experimentally induced primary amebic meningoencephalitis in mice and natura11y acquired primary amebic meningoencephalitis in humans have a similar incubation period, the disease is essentially confined to the central nervous system, similar clinical and pathological features occur and the outcome is invariable fatal (Carter, 1972; Culbertson, 1971; Duma, 1972 and Martinez et a1., 1973). A150, mice are versatile, inexpensive, easy to handle and small enough to be housed in large numbers in a small area. Culbertson et al. (1968) inoculated specific pathogen-free mice intranasally (I.N.) with E, fowleri (HB-1 strain) and observed amebic hepatitis and rhinencephatitis. Similar inoculations, intravenously (I.V.) and intraperitoneally (I.P.), showed amebae to be widely disseminated throughout the mouse. Carter (1972) studied the pathogenicity of E, fowleri administered by a variety of routes. Mice were inoculated by the I.N., I.V., I.P., intramuscular (I.M.), intragastric, subcutaneous, intrahepatic, anterior intracerebral, posterior intracerebral and intrapleural routes. Clinical symptoms and death occurred in all the mice inoculated I.N. and anterior or posterior intracerebrally. A third of the mice died following I.V. or intrahepatic inoculation. No clinical or pathological symptoms of primary amebic meningoencephalitis were found in the mice that were inoculated by the remaining routes. The flagellate stage of N, aerobia (N. fowleri) was inoculated I.N. into mice in which it produced fatal meningoencephalitis. Brain smears from the infected mice showed only the ameba stage, indicating that the flagellates reverted to amebae after I.N. inoculation. In all likelihood, it was amebae that actually invaded the host and were responsible for death of the mice (Singh and Das, 1972). Similar results were obtained when mice were inoculated I.N. with N. aerobia (N. fowleri) amebae (Singh and Das, 1970). Martinez et al. (1973) inoculated mice with amebae of two different strains of E, fowleri, (LEE-1 and CJ-1). After the onset of clinical symptoms (ruffed fur, circling, hunching), the disease progressed rapidly to death. Examination of brain tissue showed that both grey and white matter were affected and characterized by hemorrhage, edma, disintegration of neural structures with wide-spread invasion by amebae. Amebae were observed adjacent to arterioles and eapillaries. The nasal and olfactory mucosa was extensively infiltrated by motile amebae. Cerva, (l97l) inoculated mice intracerebrally and I.N. with Naegleria (Vitek strain). After intracerebral inoculation, all of the experimental mice died. Shortly before death the mice showed symptoms of infection such as reduction of activity, uneven coat, disturbed equilibrium and finally loss of coordination. The mice that were inoculated I.N. died a few days after those inoculated intracerebrally. Histological examination of both groups of mice showed necrosis of much of the brain tissue and hemorrhage of the frontal lobes and also destruction of the mucous membranes of the I.N. inoculated mice. Guinea pigs inoculated I.M. and subcutaneously with E, aerobia (fl, fowleri) exhibited generalized loss of weight and strength. Hind- quarter I.M. injections caused enlargement of the regional lymph nodes. There was no amebic involvement of the brain; however, hepatosplenomegaly, enlarged kidney and amebic lesions of the intestines did occur (Culbertson et al., 1968). Červa, (l97l) inoculated guinea pigs I.N. with high, medium and low doses of N. fowleri. Guinea pigs given the low dose developed an elevated body temperature for an extended period of time and over half of the guinea pigs died. In the two higher dose groups, a rise in body temperature was noted only a few days before death. In a similar experiment Singh and Das (l972) inoculated two guinea pigs I.N. with y, aerobia (fl, fowleri). Fatal meningoencephalitis developed soon afterwards. Phillips (l974) inoculated adult, germ-free guinea pigs I.N., intraorally, into the conjunctival sac and into skin lesions. Most of the guinea pigs inoculated I.N. died with meningoencephalitis. However, guinea pigs which were inoculated by the other routes remained in good health and were free of tissue damage at autopsy. Histological examination of the guinea pigs that had succumbed to g, fowleri infection (I.N. inoculated) showed destruction of the cerebellum, hemorrhagic meningitis, destruction of the frontal lobes, degradation of the meninges and a hemorrhagic condition of the anterior brain. Monkeys have been inoculated I.N., I.V. and intrathecally with y, fowleri. Those receiving amebae I.N. or I.V. exhibited no evidence of Naegleria infection or central nervous system involvement. Monkeys that died as a result of E, fowleri inoculation intrathecally developed extensive lesions in the cerebellum with only small hemorrhages in the cerebrum. Amebae were isolated from the brains, spinal cords, lungs and liver. The monkeys that survived intrathecal inoculation exhibited fever, anorexia, leukocytosis, elevated levels of serum enzymes, and varying degrees of central nervous system involvement (Wong et al., l975). Naegleria fowleri (HB-l strain) amebae have been injected into the marginal ear vein of adult rabbits. Rabbits that died exhibited extensive brain and liver damage (Culbertson et al., l968). Investigators have concluded that the natural route of invasion for y, fowleri is from the nasal mucosa through the cribiform plate and into the brain. Therefore, the logical way to inoculate experimental animals would be by I.N. installation. Unfortunately, it is often difficult to administer a consistently accurate dose I.N. The reasons are that (1) when under ether anesthesia the mice tend to sneeze out a portion of the inoculum, (2) some of the inoculum may remain on the external nares and (3) a portion of the inoculum may flow into the nasopharynx and be swallowed or, even worse, be aspirated and contribute to possible pneumonia. So, although it is possible to calculate the dose given, it is difficult to determine the number of amebae retained by the host. One way to avoid losing amebae and yet obtain results similar to I.N. installation would be to inoculate the mice I.V. The lateral tail veins are readily accessible for inoculation, the entire calculated inoculum is retained by the mouse and hematogenous spread carries amebae to the brain. The aim of my thesis is to examine the course of infection for mice inoculated I.V. with N. fowleri. The overall results of this study are that mice inoculated I.V. with N. fowleri died from meningoencephalitis similar to that observed for mice inoculated I.N. Although amebae were detected in liver, lung, spleen and kidney, pathological involvement of these tissues appeared to be minimal.

In this literature review I will briefly describe the general biology of transferable antibiotic resistance in bacteria, the genetic elements involved (plasmids) and several specific plasmid associated phenotypes. This will be followed with a review of the general biology of Bacteroides, the information known concerning its plasmid reservoir and finally a review of the information documenting the emergence of transferable clindamycin resistance in Bacteroides. The latter aspect represents the central focus of research described in this thesis.

Within the past decade, there has been increasing evidence which suggests that herpes simplex virus type two (HSV-2) is associated with cervical carcinoma. Because cancer of the cervix ranks as the second most common malignant disease of women, with 35 thousand new cases and 10,000 deaths per year, further investigation is warranted to determine if there is a causal relationship (Goldberg, 1976). Currently, most of the evidence which correlates HSV-2 with cancer of the cervix has been, for the most part, indirect: (i) there is an increased incidence of cervical anaplasia in women with cytologically detectable genital herpes infection, (ii) many patients with cervical carcinoma have a high titer of specific neutralizing antibody to HSV-2, (iii) HSV-2 has been isolated from a cell culture derived from one carcinoma in situ (Aurelian, 1976), (iv) HSV-2 membrane antigens were detected in a small percentage of cells from a culture not yielding infectious Virus. In addition to the correlative studies, the transforming capacity of HSV-2 for human embryonic cells has been clearly demonstrated in vitro, and (vi) hamster cells transformed in vitro by the virus have oncogenic potential when injected into hamsters (Rapp and Reed, 1976). Lack of more direct evidence in relation to human carcinoma, however, such as the inability to demonstrate hybridization of HSV-2 nucleic acid with that in the cervical carcinoma cell has left the issue of HSV-2 and cervical carcinoma cancer the subject of considerable controversy (zurHausen, 1976).

The cause(s) of arthritis and many other connective tissue diseases are largely unknown. An infectious agent, either bacterial or viral, has not been unequivocally isolated. Theories on the pathogenesis of arthritis abound. Many of these theories implicate the immune system as the problem. The early information on animal arthridites implicates retained bacterial cell wall antigens as the basis of the arthritis seen. How-ever, numerous types of antigens, both complex polymers and simple proteins, have been shown to persist in immune animals. The antigens persist in the lymph nodes and spleens playing a role in mediating antibody feedback mechanisms. Antigens also persist in collagenous tissues of the knees and feet of these animals. The role of retained antigen at these sites is unknown. It is possible to induce arthritis in immune animals by administering antigen in the joint. Drawing from the information presented, I chose to explore the role of retained antigen on collagenous tissue in the pathogenesis of rheumatic diseases. This project is the continuation of work begun by J. G. Tew, T. E. Mandel, G. A. Miller, and A. W. Burgess. Through their research, the groundwork of antigen retention on the flexor and extensor tendons of immune mice was reported.

Stem cell factor (SCF) is a recently characterized hematopoietic growth factor capable of stimulating the proliferation and differentiation of many hematopoietic cells, including the mast cell. We have cloned the gene encoding SCF from a cDNA library prepared from NIH 3T3 fibroblasts, and have characterized the ability of recombinant SCF to induce the development of mast cell-committed progenitors (MCCP), found in the mesenteric lymph nodes of mice infected with Nippostrongylus brasiliensis (Nb-MLN), but not naive mice. We have also examined Schwann cells, mast cells, and reproductive tissues for their ability to produce SCF. We have found that recombinant SCF alone can promote the formation of mast cell colonies from MCCP in methylcellulose. SCF affected not only MCCP proliferation, but could also induce their differentiation to connective tissue phenotype mast cells. SCF was also capable of generating mast cell colonies from precursors found in the peritoneal cavity of naive mice. Unexpectedly, infection of mice with Nippostrongylus caused a loss of the precursors from the peritoneal cavity by 14 days after infection. Mast cells are found near Schwann cells, and increases in mast cells have been noted concomitant with Schwann cell neoplasia, and nerve damage and repair. Schwann cells from several sources were shown to produce mRNA for SCF, and the protein was also detected on the cell surface by immunofluorescent staining, or as a secretable product in cell-conditioned supernatant. Schwann cells were also analyzed for the ability to produce the receptor for SCF, c-kit. Immunofluorescent staining and polymerase chain reaction (PCR) analysis has shown that a human malignant schwannoma expresses c-kit, while normal human Schwann cells as well as SV-40 transfected, primary neonatal and primary adult rat Schwann cells do not. Thus normal production of SCF could form an autocrine growth loop in some Schwann cell neoplasias which aberrantly express c-kit. Lastly, rat and human reproductive tissues were examined for expression of the SCF gene, using PCR and northern analysis. We found that SCF is expressed in a differential manner by various maternal and fetal tissues during pregnancy and the menstrual cycle. While rat tissues contained easily detectable SCF mRNA, human tissues appeared to express this gene in a lower quantity.

The objective of this study was to determine whether or not salinomycin, a drug with proven effectiveness against intestinal sporozoan parasites, has any effect on the survival time of mice infected with P. berghei.

Filoviruses cause sporadic outbreaks of highly lethal hemorrhagic fever throughout central Africa. Virus entry is mediated by the sole viral glycoprotein, GP. Furthermore, GP is the main target for neutralizing antibodies. Thus, a better understanding of GP and its functions is critical for the development of antivirals and vaccines. GP contains a high number of N- and O-linked glycans, which shield the majority of the protein. These glycans are required for cell surface interactions with C-type lectins that mediate internalization of the virus. We found that GP1, but not GP2, N-linked glycans were required for efficient entry into cells expressing the C-type lectins: L-SIGN, DC-SIGN, and LSECtin expressing cells, but O-linked glycans were sufficient for ASGPRI- and hMGL-dependent entry. However, filoviruses also utilize phosphatidylserine (PS) receptors, which bind PS in the viral membrane, to mediate entry into host cells. We found that all N-linked glycosylation sites in GP1 could be mutated without significant impact on expression. Furthermore, removal of all N-linked glycans increased entry into a PS receptor-dependent cell line and primary murine macrophages. These results correlated with an increase in sensitivity to proteolysis, which is required within the late endosome/lysosome to expose the receptor-binding domain. Surprisingly, removal of N-linked glycans that directly shield the receptor-binding domain did not allow for binding to the intracellular receptor, NPC1. Thus, proteolytic removal of heavily glycosylated domains within the late endosome/lysosome exposes critical receptor-binding residues that are masked by polypeptides and not N-linked glycans. Furthermore, removal of the conserved N-linked glycan on the heptad repeat 1 region in GP2 led to an increase in entry. Conversely, removal of the conserved N-linked glycan on the heptad repeat 2 region decreased entry. Removal of either glycan resulted in a decrease in entry mediated by protease-treated GP. Together, these results suggest N-linked glycans on GP2 are involved in controlling fusion. Interestingly, removal of N-linked glycans masking conserved regions of GP led to a significant increase in convalescent antibody-mediated neutralization. Overall, these results indicate that there is an evolutionary trade-off that results in a decrease in entry efficiency in order to protect virus from the immune system. Analysis of entry mediated by multiple species of ebolavirus indicated that the residue occupying position 95 is a critical determinant of entry. For Ebola virus (EBOV) GP, Sudan virus (SUDV) GP, and Bundibugyo (BDBV) GP, a lysine at position 95 imparts dependence on the cysteine protease cathepsin B. However, a glutamine at this position alleviates this dependence and is found in some early isolates of SUDV. Furthermore, cathepsin B dependence inversely correlated with an increase in sensitivity to protease-mediated degradation of GP. Mutation of K95 to a glutamine in EBOV GP and BDBV GP led to decreased sensitivity to NPC1 and voltage-operated calcium channel inhibitors. Conversely, mutation of the Q95 to a lysine in SUDV GP decreased sensitivity to NPC1 inhibitors and had no impact on voltage-operated calcium channel inhibitors. However, all proteins regardless of the residue at position 95 required NPC1 for entry. Together these results indicate that a single amino acid polymorphism in GP of ebolaviruses has dramatic impacts on entry factor dependence, suggesting potential differences in entry pathways.

No description available.

No description available.