Restoring ciliary function to differentiated primary ciliary dyskinesia cells with a lentiviral vector.

Primary ciliary dyskinesia (PCD) is a genetically heterogenous autosomal recessive disease in which mutations disrupt ciliary function, leading to impaired mucociliary clearance and life-long lung disease. Mouse tracheal cells with a targeted deletion in the axonemal dynein intermediate chain 1 (Dnaic1) gene differentiate normally in culture but lack ciliary activity. Gene transfer to undifferentiated cultures of mouse Dnaic1(-/-) cells with a lentiviral vector pseudotyped with avian influenza hemagglutinin restored Dnaic1 expression and ciliary activity. Importantly, apical treatment of well-differentiated cultures of mouse Dnaic1(-/-) cells with lentiviral vector also restored ciliary activity, demonstrating successful gene transfer from the apical surface. Treatment of Dnaic1(flox/flox) mice expressing an estrogen-responsive Cre recombinase with different doses of tamoxifen indicated that restoration of ∼20% of ciliary activity may be sufficient to prevent the development of rhinosinusitis. However, although administration of a ß-galactosidase-expressing vector into control mice demonstrated efficient gene transfer to the nasal epithelium, treatment of Dnaic1(-/-) mice resulted in a low level of gene transfer, demonstrating that the severe rhinitis present in these animals impedes gene transfer. The results demonstrate that gene replacement therapy may be a viable treatment option for PCD, but further improvements in the efficiency of gene transfer are necessary.

The individual host, a unique evolutionary island for rapidly dividing parasites: a theoretical approach.

It is hypothesized that rapidly dividing parasites producing high parasitemias within an individual host are in different environmental settings. It is further suggested that these infrapopulations experience the drastic environmental changes of free-living forms in an island environment and, that in chronically infected animals, the environmental conditions will over time select the parasites best suited to grow in their changing habitat. Evidence is presented to demonstrate that the host environment does change during an infection with the African trypanosomes and, that with time, each host becomes environmentally unique. Data are also provided to show that parasites cloned from different hosts are phenotypically different and are assumed to be genetically different as well. The evidence provided is consistent with the hypothesis that each individual host provides a unique habitat in which selection occurs, and that the rapidly dividing protozoans, such as the African trypanosomes and plasmodia are continuously evolving in the individual host.

Use of sodium butyrate to enhance production of retroviral vectors expressing CFTR cDNA.

Previously, we constructed a retrovirus vector (LCFSN) for transduction and expression of the cDNA encoding the normal human cystic fibrosis transmembrane conductance regulator (CFTR). The titer of virus from amphotropic packaging cells producing the LCFSN vector was low (10(3)-10(4) infectious units/ml). In an attempt to increase virus production, we used sodium butyrate (NaB) to treat murine retrovirus packaging cells producing this vector. NaB treatment increased the production of LCFSN from between 20-fold to greater than 1,000-fold, depending upon the producer clone, thereby resulting in virus titers up to about 1 x 10(7) infectious units/ml. This induction of virus titer could be accounted for, at least in part, by an increase in steady-state levels of full-length vector RNA within the producer cells. With some clonal producer cell lines, lowering the temperature of the virus harvest in combination with NaB treatment resulted in an apparent synergistic increase in virus production. The production of retrovirus vectors containing genes other than CFTR could also be increased by NaB treatment, although the enhancement in titer was modest (2-fold to 10-fold). The increase in virus production was not accompanied by an induction of replication-competent helper virus. NaB treatment also increased the transient production of retroviral vectors following DNA-mediated transfection into packaging cells such that virus titers of greater than 10(6) infectious units/ml could be readily attained.

The inheritance of factors controlling resistance in mice infected with Trypanosoma brucei rhodesiense.

In crosses between 2 recombinant inbred strains of mice (B x H-2 and B x H-14), resistance to infection with Trypanosoma brucei rhodesiense as measured by survival time is suggested to be controlled by a dominant gene(s). In prior studies using the same clone of trypanosomes, but a different set of inbred mouse strains, it was demonstrated that resistance in H-2 congenic mice was a recessive trait. This work suggests that in mouse trypanosomiasis, the number of genes involved in resistance and their dominant or recessive nature will vary between different inbred mouse strains. There was a statistically significant difference between the survival times of animals with high or low antibody anti-trypanosome titers. Differences in survival time were not correlated with the height of the first parasitemia. There was, however, a strong negative correlation between the number of trypanosomes at the second peak in parasitemia and survival time. It is also suggested that the extent to which the host is immunosuppressed early in infection determines the ability to control the later peaks in parasitemia, and, therefore, survival time.

Mechanism of lysis of Trypanosoma brucei gambiense by human serum.

Resistance to lysis by human serum (HS) is an important parameter used to distinguish Trypanosoma brucei brucei from both Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Neither the exact nature of the trypanolytic factor (TLF) nor the mechanism of action by which HS lyses susceptible trypanosomes is well understood. This report tries to elucidate the role played by the variable surface glycoprotein (VSG) coat and trypanosome surface-related processes in the mechanism of HS lysis of HS-sensitive (HSS) and HS-resistant (HSR) trypanosomes. Procyclic forms of T. brucei gambiense transformed from either HSS or HSR bloodstream stages were found to be HSR. These procyclic forms were shown to have lost their VSG coat. However, the addition of excess soluble VSG from HSS trypanosomes did not block lysis of HSS trypanosomes. Human serum lysis was significantly inhibited if the trypanosomes were incubated with membrane stabilizers, i.e., including cytochalasins (B, D, and E specifically), zinc acetate, vinblastine, and benzyl alcohol, or with the lysosomotropic agents ammonium chloride and chloroquine. The inhibition exerted by these compounds was always reversible. The results in this report, taken together, strengthen the hypothesis that the lytic factor interacts with and moves along the trypanosome surface to be internalized eventually.

Characterization of human serum-resistant and serum-sensitive clones from a single Trypanosoma brucei gambiense parental clone.

A protocol was developed to select clones of Trypanosoma brucei gambiense having different levels of resistance to normal human serum. Human serum-resistant clones were selected from a single parental clone by continuous serum treatment of infected immunosuppressed mice. Human serum-sensitive revertant clones were also obtained by continuous passage of resistant clones in immunosuppressed mice but without human serum pressure. It has been demonstrated that our trypanosome clones express distinct but stable levels of resistance. The variant antigenic type of each clone was characterized serologically and by 1-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. After selective pressure with human serum, variant antigen-type differences always occurred among clones in which different human serum susceptibilities were found. The work reported here demonstrates that in our T. brucei gambiense immunosuppressed mouse model there is a predictable association between variant antigen type and human serum resistance.

Trypanosoma brucei rhodesiense: the inhibition of HL-60 cell growth by the African trypanosomes in vitro.

Trypanosomiasis is of major public health importance in Africa where the disease affects man and livestock. In order to explore the underlying mechanisms of pathogenesis in African trypanosomiasis, we studied the inhibition of host cell (human promyelocytic HL-60 cells) growth by Trypanosoma brucei rhodesiense using an in vitro system. This inhibition was not due to changes in pH or nutritional depletion of the culture medium by the trypanosomes as inhibitory activity was still observed in cultures that had been supplemented with glucose or fresh culture medium. Our study suggests that the African trypanosomes produce a soluble factor which inhibits the growth of HL-60 cells. This growth inhibitor does not appear to kill the HL-60 cells as determined by the trypan blue dye exclusion test. The production of this factor does not require host cell contact nor does it require a host cell cofactor. The trypanosome growth inhibitor is strictly a trypanosome product. Estimation of the molecular weight of the trypanosome growth inhibitor with Amicon filters revealed that the factor is greater than 30,000 Da in size. Protease and heat treatment of the factor resulted in the depletion of inhibitory activity. These results indicate that the African trypanosomes produce a large-molecular-weight protein growth inhibitory factor which could play a role in the pathogenesis of the disease.

Relationship between human serum trypanocidal activity and host resistance to the African trypanosomes.

Results reported here show that humans have various levels of trypanocidal activity in their sera. This difference appeared stable when different samples were taken from the same individuals over time. It was not possible to account for the variability between individuals by obvious differences in health, nutrition, or living habits. In addition, the trypanocidal titers did not vary significantly when stored for various lengths of time at -70 C. To examine the relationship between the titer of trypanocidal activity in a host and the degree of human serum resistance of the challenge trypanosome inoculum, mice (C57BL/6J) were pretreated with various amounts of different human serum and then infected with clones having different degrees of resistance to human serum. It was demonstrated that host susceptibility to an African trypanosome infection depends upon 2 variables: the level of trypanocidal activity in individual human serum and the degree of human serum resistance of individual clones of African trypanosomes. Based upon the animal model presented here, it is hypothesized that this relationship is under selective evolutionary pressure and will influence the susceptibility of animals in endemic areas as well as the transmission of human trypanosomiasis.

A survey for a trypanocidal factor in primate sera.

The sera of 21 different species of primates were surveyed for the presence of a trypanocidal factor to a monomorphic human serum-sensitive clone of Trypanosoma brucei gambiense (T.b.g.); human, gorilla, baboon (2 species), and the mandrill were found to contain this factor. The factor in all the sera is in the high density lipoprotein (HDL) fraction, and has similar modes of biological action. It has been shown that the human and gorilla trypanocidal factor share cross-reactive antigenic epitopes, but do not share similar cross-reactive epitopes with the baboon and mandrill factor. There was no relationship between the presence or absence of this factor and the primate's position on the phylogenetic tree. In addition, there was also no obvious correlation between the animals' preferred diet, and the presence or absence of trypanocidal activity. The evidence to date suggests that only African ground-dwelling primates that live in tsetse endemic areas contain the trypanocidal factor. It is assumed that this factor is involved in resistance of these primates to T.b.b. We believe that the host has developed trypanocidal substances as a result of selective evolutionary pressure by the African trypanosomes.

Nature of the trypanocidal factor in human serum.

The chemical nature of the trypanocidal factor in human serum was investigated. The results show that although the trypanocidal factor is contained within the high density lipoprotein (HDL) fraction of human serum, it is apparently not one of the major apolipoproteins of the HDL complex such as apolipoprotein A-I, A-II, or apolipoprotein B. The factor would appear to be a minor component of the HDL fraction whose chemical nature is still uncertain.

Mechanism of long slender (LS) to short stumpy (SS) transformation in the African trypanosomes.

The transformation of the long slender to the short stumpy stages of the African trypanosomes is an essential part of the trypanosome life cycle. Four possible mechanisms which could control this event have been investigated. It has been shown that (a) the dividing long slender to non-dividing short stumpy transition is not a programmed event in the trypanosome life cycle; nor (b) would it appear to be initiated by some form of cell to cell contact inhibition of growth. In addition, evidence is presented which would suggest that (c) the transition is not started by the depletion of a critical growth nutrient from the environment during the growth of the trypanosomes. The last possibility (d) considered is that during trypanosome growth, a growth inhibitor-short stumpy inducer accumulates in the trypanosomes' environment. Evidence is presented which shows that plasma from infected animals can inhibit the incorporation of thymidine by the trypanosomes. These data are consistent with the suggestion of an exogenous growth inhibitor accumulating during the infection.

African trypanosomes: inheritance of factors involved in resistance.

The C3HeB/FeJ mouse strain has a shorter survival time and is therefore more susceptible to a Trypanosoma brucei rhodesiense infection than the B10.BR/SgSnJ strain. The work reported here demonstrated that survival time is inherited as a recessive trait, whereas the ability to produce antibody to the first variant antigen population is inherited as a dominant trait. It was therefore not possible to correlate survival time with the ability to produce antibody in the F-1 and F-2 offspring. Both characteristics appeared to be multigenic. In addition, it was not possible to link the ability of an animal to control its early parasitemia, or its change in hematocrit, with either antibody production or survival time. The work strongly suggests that the increased survival time of the B10.BR/SgSnJ mouse is due at least partially to nonspecific but unidentified factors which do not segregate with VSG-specific immune responses. These nonspecific factors could include differences in susceptibility to toxic trypanosome catabolites.

Changes in albumin levels in blood and urine of Microtus montanus chronically infected with Trypanosoma brucei gambiense.

Serum albumin and glucose concentrations and urinary excretion of alpha-keto acids and proteins were determined in samples obtained throughout a chronic Trypanosoma brucei gambiense infection in Microtus montanus. An increase in urinary excretion of alpha-keto acids and proteins during the terminal stage of disease was accompanied by a decrease in serum glucose concentration. This terminal hypoglycemia reflected a depletion of liver glycogen in most animals. In contrast (and the major focus of this study) serum albumin concentration was decreased by the second week of infection and in the final sample obtained was less than 50% of that measured in preinfection samples. Female animals survived approximately 1 wk longer than males and were less susceptible during the acute phase of disease. This relative resistance was most likely due to the fact that female animals were relatively more efficient in limiting parasitemia during the first week of infection. The similarity between humans and voles in terms of protein and alpha-keto acid excretion and changes in serum concentrations of glucose and albumin during trypanosome infection further validate the use of Microtus as an experimental model for trypanosomiasis in humans.

Growth of pleomorphic Trypanosoma brucei rhodesiense in irradiated inbred mice.

It was shown that irradiation (650 rad) of 7 inbred strains of mice did not block the ability of Trypanosoma brucei rhodesiense to transform from the long slender (LS) to the short stumpy (SS) form or alter the plateau in parasitemia. In addition, it was observed that significant differences in parasitemia levels, in the rate of transformation from the LS to the SS form, as well as in the survival times occurred between the irradiated C3HeB/FeJ and several of the other strains. These differences in the nonspecific ability to control parasitemia appeared to be characteristic for each inbred strain of mice. The resistant strains generally had lower parasitemia than the susceptible strains. However, it was also shown that there is not a one-to-one correlation between the innate ability of a mouse strain to control its initial parasitemia, and the strain's ability to clear the parasitemia or increase its survival time. It was therefore concluded that the hypothesis which states that the ability of an animal to increase nonspecifically the rate of transformation, and therefore to lower the parasitemia, allowing intact animals to respond immunologically and survive longer is either incorrect or incomplete. The results further show that the ability of mice to clear their initial parasitemia by an antibody response is not necessarily correlated with their survival time. Therefore, this study suggests that factors other than an antibody response and the nonspecific control of parasitemia are important in resistance.

Immune response to minor variant antigen types (VATs) in a mixed VAT infection of the African trypanosomes.

It has been shown that soon after the onset of acute infection with Trypanosoma brucei gambiense, mice are able to detect immunologically small numbers of minor variant antigen types (VATs) within the population. However, in more longstanding infections, considerably larger populations of minor VATs are required to stimulate an effective immune response. As a result, larger populations of minor VATs evade immune detection and, following a decrease in parasitaemia, become part of the relapse population. We hypothesize that the development of immunosuppression increases the effectiveness of antigenic variation as an escape mechanism.

Further biochemical characterization of chronic Trypanosoma brucei gambiense-Microtus montanus infection.

Tyrosine aminotransferase (TAT), glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), and alkaline phosphatase (ALP) were measured in the serum and livers of Microtus montanus infected with Trypanosoma brucei gambiense. Only liver TAT and serum ALP showed significant changes. In addition, blood glucose, pyruvate and lactate, and liver glycogen levels were assayed. All four compounds showed significant changes, strongly suggesting increased glycogen mobilization and increased catabolic activity. Interestingly, the serum ketone levels were very low and no significant changes were observed. These chronically infected animals had an organic aciduria in which pyruvate, lactate, beta-hydroxybutyrate, alpha-ketoglutarate, phenylpyruvate, and p-hydroxyphenylpyruvate were significantly increased. The possible significance of these observations is discussed.

Multiple alpha-keto aciduria in Microtus montanus chronically infected with Trypanosoma brucei gambiense.

Microtus montanus chronically infected with a monomorphic strain of Trypanosoma brucei gambiense excreted in urine greatly elevated quantities of not only the aromatic alpha-keto acids, phenylpyruvic and 4-hydroxyphenylpyruvic acids, but also two aliphatic alpha-keto acids, pyruvic and alpha-ketoglutaric acids. Elevated keto acid excretion began approximately midway through infection and quantities remained elevated until death. Daily keto acid excretion did not correlate with daily parasitemia. Thus, a large metabolic disturbance exists in laboratory animals infected with African trypanosomes. The multiple alpha-keto aciduria potentially contributes to the pathogenesis of chronic African trypanosomiasis.

The ecology of antigenic variation.

A detailed molecular analysis using recombinant DNA technologies is extremely important to our understanding of the phenomena of antigenic variation in the African trypanosomes; however, by itself, it may not completely explain antigenic variation as it occurs in vivo. Several laboratories have demonstrated the ability of one variant population to replace another in vivo as well as the presence of heterogeneous populations of trypanosomes within an individual animal. These two phenomena do not permit us to explain antigen variation solely on the basis of the molecular regulation of variant antigen expression. In addition to studies in molecular biology, it will be necessary to define clearly the differences in growth rates of variant populations and the role of competition between these variants in a single anatomical site. It will also be necessary to determine the influence of various physiological environments on growth rates and the competition between the different variants of a single repertoire. It is concluded that the phenomenon of antigenic variation is a complex problem in ecology and population dynamics as well as molecular regulation. This paper is designated to examine a variety of the ecological parameters presumably involved in antigenic variation.

Phenylalanine metabolism in Microtus montanus chronically infected with Trypanosoma brucei gambiense.

1. Alterations in phenylalanine metabolism were observed in Microtus montanus infected with Trypanosoma brucei gambiense. 2. Reductions were found in the incorporation of phenylalanine into free amino acid pools and into proteins of the liver and other organs of infected animals. 3. Increased quantities of phenylpyruvate, 4-hydroxyphenylpyruvate and pyruvate were found in the urine of infected Microtus. 4. There appeared to be very limited conversion of phenylalanine to tyrosine in infected animals. This suggested that either phenylalanine transaminase activity was greatly elevated in proportion to phenylalanine hydroxylase activity; or that phenylalanine hydroxylase activity was actually reduced during the infection.