Correlation of virulence, lung pathology, bacterial load and delayed type hypersensitivity responses after infection with different Mycobacterium tuberculosis genotypes in a BALB/c mouse model.

One of the most intriguing aspects of tuberculosis is that the outcome of an infection with M. tuberculosis (TB) is highly variable between individuals. The possibility of differences in virulence between M. tuberculosis strains or genotypes has only recently been studied. There is evidence of multifactorial genetic predisposition in humans that influences the susceptibility to tuberculosis. A better understanding of differences in virulence between M. tuberculosis genotypes could be important with regard to the efforts at TB control and the development of improved antituberculosis vaccines. Survival, lung pathology, bacterial load and delayed type hypersensitivity (DTH) responses of BALB/c mice after intratracheal infection with any of 19 different M. tuberculosis complex strains of 11 major genotype families were studied. The results indicate that among genetically different M. tuberculosis strains a very broad response was present with respect to virulence, pathology, bacterial load and DTH. 'Low'-responders were the H37Rv, Canetti, Beijing-1 strains, while Beijing-2,3, Africa-2 and Somalia-2 strains were 'high'-responders. A severe pathological response correlates with a high mortality and a high CFU counts in lungs, but poorly with the degree of the DTH response.

Chlamydophila abortus infection in a pregnant woman associated with indirect contact with infected goats.

Reported here is the case of a pregnant woman who developed a severe Chlamydophila abortus infection after indirect contact with infected goats resulting in preterm stillbirth. The woman fully recovered after treatment with doxycycline. In the goat herd with which her husband worked Chlamydophila abortus was actively circulating, as shown by positive serology. When pregnant women present with rapidly worsening influenza-like illness, special attention should be given to possible contact (direct or indirect) with animals when recording the anamnesis. Pregnant women, especially those who live in rural areas, should generally be made aware of the risks of zoonotic diseases and how to avoid them.

Pathogenesis of poliovirus infection in PVRTg mice: poliovirus replicates in peritoneal macrophages.

The pathogenesis of poliovirus infection, responsible for the induction of a poliovirus-specific mucosal immune response following intraperitoneal (i.p.) inoculation of virus in mice transgenic for the poliovirus receptor (PVRTg mice), was studied. Following inoculation of poliovirus, replication was determined by increase in virus titre (TCID(50)) and by PCR of poliovirus-specific negative-strand RNA in peritoneal macrophages, mesenteric lymph nodes, Peyer's patches, duodenum, brain, kidney and liver. The presence of poliovirus antigens in several cell types was detected by immunolabelling. It was demonstrated that poliovirus replicated in the peritoneal macrophages of PVRTg mice, since the virus titre in peritoneal cells was increased compared to the titre in the inoculum. Negative-strand RNA was detected in these cells and most of the poliovirus-immunostained cells had the morphology of macrophages and expressed the macrophage-specific markers CD86 and M1/70 on their surface. Furthermore, in peritoneal lavage, poliovirus was also present in CD19(+) B cells, but not in dendritic or T cells. Moreover, poliovirus was detected in macrophage-like cells in the lamina propria of the intestine, but not in epithelial cells. Replication of poliovirus in mesenteric lymph nodes, Peyer's patches and brain was followed by excretion of virus in the faeces. This suggests that the virus is transported due to migration of macrophages from the peritoneal cavity to mesenteric lymph nodes and the lamina propria of Peyer's patches. It is likely that this route is responsible for the induction of virus-specific IgA in the gut.

In vitro processing and presentation of a lipidated cytotoxic T-cell epitope derived from measles virus fusion protein.

Lipopeptidic formulations have been described as efficient activators of cytotoxic T lymphocytes (CTL). To better understand the pathway via which lipopeptides reach the MHC class I molecules we studied the intracellular processing and presentation of a measles virus-derived CTL epitope, to which a palmitoyl moiety was added synthetically. The palmitoyl group was conjugated to the N-terminus either directly or via a spacer sequence. The use of single or double fluorescent-labeled lipopeptides allowed the visualization of intracellular processing of these antigens using confocal microscopy. Our data indicate that the spacer composition influences internalization of the conjugate into the cell, proteasomal degradation, translocation into the ER by the transporter associated with antigen processing (TAP), and the intracellular trafficking of lipopeptides.

Lipid-coated polyplexes for targeted gene delivery to ovarian carcinoma cells.

A nonviral gene delivery vector has been developed in our laboratory based on the cationic polymer, poly(2-(dimethylethylamino)ethyl methacrylate) (p(DMAEMA)). p(DMAEMA)-based polyplexes have been successfully used for the transfection of OVCAR-3 cells in vitro. However, these polyplexes were unable to transfect OVCAR-3 cells growing in the peritoneal cavity of nude mice after intraperitoneal administration, which could be ascribed to inactivation by components (including hyaluronic acid) present in the tumor ascitic fluid. The present work aimed at (a) protecting p(DMAEMA)-based polyplexes against destabilization or inactivation by polyanions such as hyaluronic acid present in tumor ascitic fluid and (b) enhancing cellular uptake of the protected p(DMAEMA)-based polyplexes by targeting with antibody Fab' fragments. To fulfill these requirements, we have developed a detergent removal method to coat polyplexes with anionic lipids. With this method, spherical particles of approximately 125 nm, which were protected from destabilization by polyanions, were obtained. More importantly, the transfection efficiency of lipopolyplexes was unaffected in the presence of hyaluronic acid, indicating that lipid coating of polyplexes protects against destabilization by hyaluronic acid. By conjugating antibody Fab' fragments directed against the epithelial glycoprotein-2 to the lipidic surface of these lipopolyplexes, target cell-specific transfection of OVCAR-3 cells could be obtained in vitro.

The adventitia of atherosclerotic coronary arteries frequently contains Chlamydia pneumoniae.

The presence of Chlamydia pneumoniae in the human arterial system has mainly been determined in atherosclerotic plaque, whereas the adventitia has remained relatively unexplored. We assessed the presence of C. pneumoniae in all three vessel wall layers of coronary (n=72) and brachial (n=48) arteries in relation to local atherosclerosis. Immunohistochemical staining of C. pneumoniae was observed in plaque and adventitia. Cells stained for C. pneumoniae were detected in the same areas as cells stained for macrophages in adjacent sections. C. pneumoniae staining in the adventitia was associated with the extent and severity of atherosclerosis. Coronary sections with C. pneumoniae staining in both adventitia and plaque more often contained advanced atherosclerosis than sections with staining only in the adventitia. Staining was observed more often in the coronary artery than in the brachial artery (24/72 vs. 5/48 and 51/72 vs. 8/48 for plaque and adventitia, respectively, P=0.004 and P<0.001). PCR confirmed the presence of C. pneumoniae DNA in the adventitia. In summary, the adventitia of atherosclerotic coronary arteries frequently contains C. pneumoniae that seems to be located within macrophages. These results might indicate a possible route for infected circulating macrophages to home into atherosclerotic lesions in the artery via vasa vasorum.

UVA exposure affects UVB and cis-urocanic acid-induced systemic suppression of immune responses in Listeria monocytogenes-infected Balb/c mice.

Ultraviolet radiation can inhibit immune responses locally as well as systemically. Such effects have been measured in animals and humans exposed to ultraviolet B (wavelength 280-315 nm) (UVB) and ultraviolet A (315-400 nm) (UVA). The precise wavelength dependence is important for the identification of possible molecular targets and for assessments of risk of different artificial UV sources and solar UV. In such analyses, it is commonly assumed that radiation energy from each wavelength contributes to the effect independent of the other wavelengths. Here we show that this assumption does not hold good. In the present study, it was investigated whether exposure to broadband UVA or longwave ultraviolet A 1 (340-400 nm) (UVA 1) prior to the standard immunosuppressive UVB protocol might modulate the immunosuppressive effects induced by UVB. Preexposure to broadband UVA or longwave UVA 1, 1 day prior to the standard immunosuppressive UVB protocol, inhibited the UVB-induced suppression of delayed type hypersensitivity (DTH) to Listeria monocytogenes significantly. This effect was not associated with restoring the number of interleukin (IL-12)-positive cells in the spleen. Since isomerization of trans-urocanic acid (UCA) into the immunosuppressive cis-UCA isomer plays a crucial role in UVB-induced immunomodulation, in a second set of experiments it was investigated whether immunosuppression induced by cis-UCA might also be downregulated by preexposure to UVA. Animals were exposed to broad-band UVA or longwave UVA 1 prior to application of an immunosuppressive dose of cis- or trans-UCA as a control. Both UVA and UVA 1 appear to inhibit the cis-UCA-induced systemic immunosuppression (DTH and IL-12) to L. monocytogenes. These studies clearly show that UVA radiation modulates both UVB and cis-UCA-induced immunomodulation. In general, our studies indicate that both broadband UVA and longwave UVA 1 could induce modulation of UVB and cis-UCA-induced immunomodulation. As sunlight contains both UVA and UVB radiation the balance between these two radiations apparently determines the net immunomodulatory effect.

Distribution of Chlamydia pneumoniae in the human arterial system and its relation to the local amount of atherosclerosis within the individual.

BACKGROUND: Chlamydia pneumoniae has been suggested to play a role in the origin of atherosclerosis. We studied the prevalence of C pneumoniae at multiple locations in the arterial system within the same individual. Studying the association between atherosclerosis and C pneumoniae within the individual excludes confounding by interindividual variability. METHODS AND RESULTS: Postmortem, the presence in the intima/plaque and media of C pneumoniae membrane protein was determined by use of a C pneumoniae-specific monoclonal antibody. In 24 individuals, 33 arterial locations were studied (n=738 segments). Area stenosis was determined in adjacent cross sections. In all individuals, immunostaining of C pneumoniae was observed in >/=1 artery. The highest prevalences were observed in the abdominal aorta (67%), internal and common iliac arteries (41%), and coronary arteries (33%). The lowest prevalences were observed in the radial (0%) and cerebral (2%) arteries. Within the individual, area stenosis was larger in cross sections with immunoreactivity compared with cross sections without immunoreactivity (31.0+/-11.9% versus 14.3+/-6.1%, respectively; P:<0.001). In the individual, immunoreactivity was observed in 15+/-10% of the arteries (range, 3% to 45%). Between individuals, the percentage of arteries with immunoreactivity to C pneumoniae was associated with the average area stenosis throughout the arterial system (r(2)=0.56, P:<0.001). CONCLUSIONS: C pneumoniae was mostly observed at locations that are related to clinically relevant features. Within the individual, the distribution of C pneumoniae is associated with the distribution of atherosclerosis. The role of the microorganism in atherosclerotic disease remains to be elucidated.

Acholeplasma vituli sp. nov., from bovine serum and cell cultures.

Organisms isolated from commercial foetal bovine serum and from cell culture lines containing such serum supplements were found to consist of non-helical, non-motile, pleomorphic coccoid forms. One strain (FC 097-2T) cultivated directly from foetal bovine serum was selected for characterization. In ultrastructural examination, individual round cells lacked cell wall structures and cells varied in size, with a mean diameter of about 700 nm. However, variable numbers of cells were filterable through membranes of 300 nm. Optimum growth occurred between 30 and 37 degrees C. The organism fermented glucose, fructose and mannose, but did not hydrolyse arginine. The strain was insensitive to 500 U penicillin ml(-1) and was capable of growing in the absence of serum or cholesterol. The organism was serologically distinct from all 13 currently described species in the genus Acholeplasma and from other sterol-requiring species in the genus Mycoplasma, using growth inhibition, immunoperoxidase and immunofluorescence tests. Strain FC 097-2T was found to have a DNA G+C composition between 37.6 +/- 1 mol% and 38.3 +/- 1 mol%. The genome size was determined to be 2095 kbp. The 16S rDNA sequence of strain FC 097-2T was compared to 16S rDNA sequences of other mollicutes in nucleotide databases. No deposited sequence was found to be identical; the closest relatives were several members of the genus Acholeplasma. On the basis of these findings and other similarities to acholeplasmas in morphology and growth, the absence of a sterol requirement for growth, and similar genomic characteristics, the organism was assigned to the genus Acholeplasma. Strain FC 097-2T is designated the type strain (ATCC 700667T) of a new species, Acholeplasma vituli.

In vivo antibody response and in vitro CTL activation induced by selected measles vaccine candidates, prepared with purified Quil A components.

Semipurified Quil A and purified Quil A were used to prepare well-characterized subunit vaccine candidates against measles. Variation in the relative amounts of the measles virus (MV) fusion (F) protein, Quil A-components and lipids did not influence induction of antibody responses in mice, but had a pronounced effect on the capacity to induce cytotoxic T cell (CTL) activity of a CD8(+) MV F-protein specific human T cell clone in vitro. A characteristic MV iscom preparation based on the combined use of HPLC-purified Quil A-components QA-3 and QA-22 (QA-3/22) efficiently induced CTL activity in vitro. Comparable results were obtained by mixing beta-propiolactone inactivated MV with iscom-matrix QA-3/22 or free QA-22. On the basis of the data presented it was concluded that these three preparations are interesting MV vaccine candidates for further evaluation in pre-clinical experiments in a primate model.

Chlamydia pneumoniae in vitro and in vivo: a critical evaluation of in situ detection methods.

AIMS: There is a considerable discrepancy between data from the detection of Chlamydia pneumoniae in atherosclerotic lesions obtained by means of immunocytochemistry and data obtained using the polymerase chain reaction. This study evaluated methods for the in situ detection and assessment of the viability of C pneumoniae bacteria. METHODS: Chlamydia pneumoniae membrane protein, heat shock protein 60, and lipopolysaccharide were detected by immunocytochemistry, and genomic DNA and 16S rRNA by in situ hybridisation in paraffin wax embedded sections of cultured HEp2 cells infected with C pneumoniae and of lungs from mice infected intranasally with C pneumoniae. RESULTS: Inclusions reactive for all three antigens, DNA, and 16S rRNA were seen in infected HEp2 cells, in all positive bronchus and alveolar epithelial cells, and in some of the positive infiltrate cells in the lungs of mice up to seven days after infection. In all alveolar macrophages and in the infiltrate cells positive for antigens only, the staining pattern was granularly dispersed throughout the cytoplasm up to seven days after infection. At 21 days after infection, only this granular staining pattern was seen for antigens in infiltrate cells and macrophages in the alveoli and bronchus associated lymphoid tissue. At this time point, DNA or 16S rRNA were detected sporadically, but always as inclusion-like staining. CONCLUSIONS: Because antigens with an inclusion-like staining were detected only together with DNA and 16S rRNA, this type of staining pattern suggested the presence of viable bacteria. Thus, the granular staining pattern of antigens in the absence of staining for DNA and 16S is most likely caused by non-viable bacteria. In conclusion, these methods are suitable for the in situ detection of C pneumoniae and the assessment of its viability.

Chlamydia pneumoniae antigens, rather than viable bacteria, persist in atherosclerotic lesions.

AIMS: To evaluate the nature of the presence of Chlamydia pneumoniae or of other members of the order Chlamydiales in atherosclerotic lesions. METHODS: Consecutive sections of 13 carotid artery specimens obtained at necropsy and of C pneumoniae infected HEp2 cells were analysed using: (1) immunocytochemistry (ICC) to detect C pneumoniae membrane protein; (2) in situ hybridisation (ISH) using a polymerase chain reaction (PCR) fragment of the omp1 gene to detect C pneumoniae specific DNA; (3) ISH using an oligonucleotide probe to detect Chlamydiales specific 16S rRNA; (4) PCR to detect C pneumoniae 16S rDNA; and (5) in situ DNA nick and labelling (TUNEL) to detect fragmented DNA. RESULTS: Staining by ICC and ISH of infected HEp2 cells showed characteristic inclusions. Chlamydia pneumoniae membrane protein was demonstrated in macrophages in advanced atherosclerotic lesions (six of six), but not in fatty streaks (none of two), or normal arteries (none of five). ISH assays using both probes and PCR were all negative, indicating the absence of both specific C pneumoniae DNA and Chlamydiales specific 16S rRNA. Only after treatment with DNAse I were uniformly sized dots demonstrated by the TUNEL assay in inclusions of infected HEp2 cells. The TUNEL assay showed a similar staining pattern in macrophages in five carotid artery specimens, of which four were also positive for C pneumoniae membrane protein. Both macrophage populations were morphologically similar and were similarly distributed. CONCLUSIONS: No evidence was obtained for the involvement of other members of the order Chlamydiales in atherosclerosis. The presence of C pneumoniae antigen in the absence of DNA and 16S rRNA suggests that antigens, rather than viable bacteria, persist in atherosclerotic lesions.

Rational medium design for Bordetella pertussis: basic metabolism.

In current Bordetella pertussis media ammonium accumulates because of an imbalance in the nitrogen:carbon ratio of the substrates used, which is one of the factors limiting cell density in fed-batch cultures. The aim of this study was to map B. pertussis catabolic and anabolic capabilities, in order to design a medium that avoids ammonium accumulation, while substrates are metabolised completely. Besides the known dysfunctional glycolysis, B. pertussis also possessed a partially dysfunctional citric-acid cycle. Although ammonium accumulation was avoided by adding various carbon sources to medium with glutamate, nuclear magnetic resonance (NMR) showed excretion of acetate, acetoacetate and beta-hydroxy-butyrate, thereby reducing the biomass yield. Acetoacetate and beta-hydroxy-butyrate were also formed in Verwey, B2 and modified Stainer-Scholte medium. Electron microscopy in combination with NMR showed that cells early on in these cultures contained poly-hydroxy-butyrate (PHB) globules, which disappeared later during the culture, coinciding with the appearance of beta-hydroxy-butyrate and/or acetoacetate. No globules nor metabolite excretion was detected when lactate in combination with glutamate were used as substrates. Thus, metabolite excretion and ammonium accumulation were avoided, while the yield of 8.8 g C-mol-1 compared favourably with literature values, averaging 6.5 g C-mol-1. Optimisation of this medium for pertussis toxin production will be reported in a separate article.

Solid-phase synthesis and application of double-fluorescent-labeled lipopeptides, containing a CTL-epitope from the measles fusion protein.

The mechanism which enables lipopeptides to induce cytotoxicity is not known. By preparing fluorescent-labeled lipopeptides one might unravel the mechanism of their entry into the cell and their intracellular pathway. A method of preparing double-fluorescent-labeled peptides by solid-phase chemistry is described. As model peptides we have chosen analogs of the sequence RRYPDAVYL, which occurs in the measles fusion protein (F438-446) and is an epitope for cytotoxic T lymphocytes. The peptides Pal-K(TMR)KKKRRYPDAVK(FL)L (7) and Pal-K(FL)KKKRRYPDAVK(TMR)L (8), in which Pal is palmitoyl and K(TMR) and K(FL) are Nepsilon-carboxytetramethylrhodamine- and Nepsilon-carboxyfluorescein-labeled lysyl residues, respectively, were prepared and obtained in approximately 30% yield after purification by high-performance liquid chromatography. The fluorescence of fluorescein and tetramethylrhodamine in lipopeptide Pal-K(TMR)KKKRRYPDAVK(FL)L (7) was quenched to 98-99% due to intramolecular interaction of the labels. On incubation with trypsin (i.e. cleavage at the KKKRR-site) the fluorescence of both labels was restored. The intracellular routing of lipopeptide Pal-K(TMR)KKKRRYPDAVK(FL)L was studied with human melanoma cell line, Mel/J, which was transfected with human leukocyte antigen B*2705. It appeared that the double-fluorescent-labeled lipopeptide was able to induce antigen-specific cytotoxicity. Furthermore, preliminary confocal microscopical studies indicated that this lipopeptide is observed intracellularly.

Chlamydia pneumoniae in abdominal aortic aneurysms: abundance of membrane components in the absence of heat shock protein 60 and DNA.

In this article, we describe the results of a comparative study for the detection of Chlamydia pneumoniae in abdominal aortic aneurysm specimens of 19 patients through the use of immunocytochemistry (ICC), in situ hybridization (ISH), and polymerase chain reaction (PCR), along with the detection of cytomegalovirus (CMV) and herpes simplex virus (HSV) by ICC and PCR. C pneumoniae-specific membrane protein was detected in specimens of all 19 (100%; 95% confidence interval [CI] 82% to 100%) and of 15 (79%; 95% CI 54% to 94%) patients with monoclonal antibodies RR-402 and TT-401, respectively. Chlamydial lipopolysaccharide was detected in specimens of 15 (79%; 95% CI 54% to 94%) patients when the results of 4 different monoclonal antibodies were combined. Surprisingly, chlamydial heat shock protein 60 was not detected in any of the specimens by ICC. Furthermore, C pneumoniae DNA was not detected by ISH when a C pneumoniae major outer membrane protein gene fragment was used as probe, nor was it reproducibly detected by PCR on extracted DNA. These results may be explained either by different kinetics of degradation of the different components of C pneumoniae after infection of the vessel wall or by the involvement of other Chlamydia-like microorganisms. Coexistence of C pneumoniae antigens and HSV antigens but not CMV antigens was observed in specimens from 10 of 18 (56%; 95% CI 31% to 78%) patients by ICC. CMV and HSV DNAs were not detected by PCR. In conclusion, we have demonstrated the presence of antigens of C pneumoniae in the absence of specific DNA in abdominal aortic aneurysms, suggesting persistence of the antigens rather than a persistent infection.

A new rat model of otitis media caused by Streptococcus pneumoniae: conditions and application in immunization protocols.

Streptococcus pneumoniae (pneumococcus [Pn]) can be cultured from up to 50% of acute otitis media (AOM) effusions, and these bacteria are the most common cause of AOM-related complications. With the recent advent of antibiotic-resistant Pn strains, treatment of Pn infections may meet with serious difficulties. Prevention through vaccination, notably for the four most common occurring Pn serotypes in humans (i.e., Pn 6B, Pn 14, Pn 19F, and Pn 23F), is a helpful alternative. Testing of vaccine efficacy should occur in an appropriate animal AOM model, which is presented here. The four involved Pn serotypes are not pathogenic to the rat, which was chosen as the experimental animal for practical reasons. To induce a natural infection (i.e., ascending through the eustachian tube), the mucociliary clearance of the eustachian tube was impaired by infusing histamine into the tympanic cavity on 2 consecutive days before intranasal inoculation of the bacteria. With this simple protocol, high and reproducible infection rates, as determined with bacterial cultures, of Pn-induced AOM (approximately 70%) with the two major Pn serotypes 14 and 19F (Pn 14 and Pn 19F) were obtained, whereas lower infection rates (25 to 50%) with Pn 6B and Pn 23F were obtained. In this model, intranasal priming with pneumococci, as well as subcutaneous vaccination with Pn 14 tetanus toxoid-conjugated polysaccharide, induced a protective effect against the induction of otitis media with these bacteria. This shows that immunity to Pn 14 AOM can be induced by both mucosal and systemic presentations of antigen. In conclusion, we have developed an animal model for Pn-induced AOM, which is suitable for the evaluation of the protecting effect of immunization.

Detection of microorganisms in vessel wall specimens of the abdominal aorta: development of a PCR assay in the absence of a gold standard.

A procedure in which the "Invitrogen Easy-DNA" kit was followed by a silica-based method for the isolation of DNA was developed for extraction of PCR-inhibitor-free DNA from up to 300 mg of human vessel wall tissue. Optimally designed PCR assays were developed for the detection of at least one infected cell in this amount of tissue. Details of the procedure are given for the detection of DNA of Chlamydia pneumoniae, cytomegalovirus and herpes simplex virus type 1 and type 2 in human vessel walls. The procedure can serve as a reference method or as a gold standard when a high-performance method is needed.

Comparative immunotoxicology of ultraviolet B exposure I. Effects of in vitro and in situ ultraviolet B exposure on the functional activity and morphology of Langerhans cells in the skin of different species.

Ultraviolet (UV) B-induced morphological and functional changes in the skin of mice, rats and humans were investigated. Changes in the morphological structure of Langerhans cells (LC), the major antigen-presenting cells in the skin, using confocal laser scanning microscopy, were found in mouse and rat skin after in situ exposure to high doses of UVB radiation (FS40) (3-9 kJ/m2). Similar UVB doses failed to induce alterations in the morphological structure of human LC. Alterations in the function of epidermal cells (especially LC) were studied, using the mixed skin lymphocyte response (MSLR). In vitro UVB exposure of epidermal cells (EC), derived from the skin of the different species, revealed that low doses of UVB radiation impaired the stimulatory capacity of these cells dose-dependently; mouse epidermal cells were most UVB-susceptible, while human cells were least UVB susceptible. For suppression of the stimulatory capacity of EC after in situ UVB exposure of skin tissue, higher doses of UVB radiation than the in vitro UVB exposure were needed in all species tested. Also in this in situ set-up mouse epidermal cells were most UVB-susceptible, and human epidermal cells were least UVB-susceptible. The magnitude of differences in susceptibility for UVB-induced changes in the stimulatory capacity of EC after in situ and after in vitro exposure experiments was similar. Firstly, it may be concluded that UVB impairs the functional activity of LC at a lower dose than that which alters the morphology of these cells. Secondly, it is clear that epidermal cells, especially LC, from the skin of rodents are more susceptible to UVB than epidermal cells derived from human skin. It is important to account for these differences in susceptibility when data on the effects of UVB radiation on the immune system in rodents are extrapolated to humans.

Effects of in vivo exposure to bis(tri-n-butyltin)oxide, hexachlorobenzene, and benzo(a)pyrene on cytokine (receptor) mRNA levels in cultured rat splenocytes and on IL-2 receptor protein levels.

Analysis of cytokine (receptor) mRNA levels has been suggested to be a sensitive technique for predicting the immunomodulatory potential of drugs and chemicals. Furthermore, this type of analysis is thought to be important in unraveling mechanisms of immunotoxicity. To study these issues, male Wistar rats were exposed to the immunotoxic environmental contaminants bis(tri-n-butyltin) oxide (TBTO; 5, 20, or 80 mg/kg diet for 6 weeks), hexachlorobenzene (HCB; 50, 150, or 450 mg/kg diet for 6 weeks), or benzo(a)pyrene (B(a)P; 3, 10, 30, or 90 mg/kg body wt for 5 weeks by a daily (5 times a week) oral intubation). Spleen cells were cultured with Con A and analyzed by dot blot hybridization for IL-2, IFN-gamma, IL-2 receptor alpha-chain (IL-2R alpha; CD25), and IL-4 mRNA levels. In addition, spleen and thymus sections of TBTO-exposed animals were assayed immunohistochemically for CD25 expression. Exposure to TBTO resulted in a dose-dependent decrease in IL-2R alpha mRNA levels from 5 mg/kg, a dose-dependent increase in IFN-gamma mRNA levels from 20 mg/kg, and increased IL-2 mRNA levels at 80 mg/kg diet. Exposure to HCB resulted in a dose-dependent increase in IL-2 and IFN-gamma mRNA levels from 150 mg/kg and increased IL-2R gamma mRNA levels at 450 mg/kg diet. Exposure to B(a)P resulted in a dose-dependent increase in IL-2 and IFN-gamma mRNA levels from 10 mg/kg and increased IL-2R alpha mRNA levels at 90 mg/kg body wt. No effects were seen on IL-4 mRNA levels. Spleen and thymus sections of TBTO-exposed animals showed reduced CD25 expression from 5 mg/kg diet. These results show that (1) the correlation between altered cytokine (receptor) mRNA levels and functional endpoints is variable, depending on the type of functional endpoint tested and the compound studied, (2) these assays are among the most sensitive ones for TBTO and HCB immunotoxicity, and among the more sensitive ones for B(a)P immunotoxicity, and (3) for TBTO, these assays provide a possible clue to a mechanism for thymus atrophy, resulting from exposure to this compound: reduced IL-2R expression may impede thymocyte maturation, resulting in thymus atrophy.