Reverse transcriptase inhibitors prevent liver abscess formation during Escherichia coli bloodstream infection.

The presence of bacteria in the bloodstream is associated with severe clinical outcomes. In mice, intravenous inoculation of Escherichia coli can lead to the formation of macroscopic abscesses in the liver. Abscesses are regions of severe necrosis and consist of millions of bacteria surrounded by inflammatory immune cells. Liver abscess susceptibility varies widely across strains of mice, but the host factors governing this variation are unknown. Here, we profiled hepatic transcriptomes in mice with varying susceptibility to liver abscess formation. We found that transcripts from endogenous retroviruses (ERVs) are robustly induced in the liver by E. coli infection and ERV expression positively correlates with the frequency of abscess formation. Hypothesizing that ERV-encoded reverse transcriptase may generate cytoplasmic DNA and heighten inflammatory responses, we tested whether nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) influence abscess formation. Strikingly, a single NRTI dose administered immediately following E. coli inoculation prevented abscess formation, leading to a concomitant 100,000-fold reduction in bacterial burden. We provide evidence that NRTIs inhibit abscess formation by preventing the tissue necrosis that facilitates bacterial replication. Together, our findings suggest that endogenous reverse transcriptases drive inflammatory responses during bacterial bloodstream infection to drive abscess formation. The high efficacy of NRTIs in preventing abscess formation suggests that the consequences of reverse transcription on inflammation should be further examined, particularly in infectious diseases where inflammation drives negative clinical outcomes, such as sepsis.

Genetic and immune determinants of E. coli liver abscess formation.

Systemic infections can yield distinct outcomes in different tissues. In mice, intravenous inoculation of Escherichia coli leads to bacterial replication within liver abscesses, while other organs such as the spleen clear the pathogen. Abscesses are macroscopic necrotic regions that comprise the vast majority of the bacterial burden in the animal, yet little is known about the processes underlying their formation. Here, we characterize E. coli liver abscesses and identify host determinants of abscess susceptibility. Spatial transcriptomics revealed that liver abscesses are associated with heterogenous immune cell clusters comprised of macrophages, neutrophils, dendritic cells, innate lymphoid cells, and T-cells that surround necrotic regions of the liver. Abscess susceptibility is heightened in the C57BL lineage, particularly in C57BL/6N females. Backcross analyses demonstrated that abscess susceptibility is a polygenic trait inherited in a sex-dependent manner without direct linkage to sex chromosomes. As early as 1 d post infection, the magnitude of E. coli replication in the liver distinguishes abscess-susceptible and abscess-resistant strains of mice, suggesting that the immune pathways that regulate abscess formation are induced within hours. We characterized the early hepatic response with single-cell RNA sequencing and found that mice with reduced activation of early inflammatory responses, such as those lacking the LPS receptor TLR4 (Toll-like receptor 4), are resistant to abscess formation. Experiments with barcoded E. coli revealed that TLR4 mediates a tradeoff between abscess formation and bacterial clearance. Together, our findings define hallmarks of E. coli liver abscess formation and suggest that hyperactivation of the hepatic innate immune response drives liver abscess susceptibility.

Different SP1 binding dynamics at individual genomic loci in human cells.

Using a tamoxifen-inducible time-course ChIP-sequencing (ChIP-seq) approach, we show that the ubiquitous transcription factor SP1 has different binding dynamics at its target sites in the human genome. SP1 very rapidly reaches maximal binding levels at some sites, but binding kinetics at other sites is biphasic, with rapid half-maximal binding followed by a considerably slower increase to maximal binding. While ∼70% of SP1 binding sites are located at promoter regions, loci with slow SP1 binding kinetics are enriched in enhancer and Polycomb-repressed regions. Unexpectedly, SP1 sites with fast binding kinetics tend to have higher quality and more copies of the SP1 sequence motif. Different cobinding factors associate near SP1 binding sites depending on their binding kinetics and on their location at promoters or enhancers. For example, NFY and FOS are preferentially associated near promoter-bound SP1 sites with fast binding kinetics, whereas DNA motifs of ETS and homeodomain proteins are preferentially observed at sites with slow binding kinetics. At promoters but not enhancers, proteins involved in sumoylation and PML bodies associate more strongly with slow SP1 binding sites than with the fast binding sites. The speed of SP1 binding is not associated with nucleosome occupancy, and it is not necessarily coupled to higher transcriptional activity. These results with SP1 are in contrast to those of human TBP, indicating that there is no common mechanism affecting transcription factor binding kinetics. The biphasic kinetics at some SP1 target sites suggest the existence of distinct chromatin states at these loci in different cells within the overall population.

Impact of the difference in diagnostic criteria for adolescent polycystic ovary syndrome excluding polycystic ovarian morphology.

AIM: Exclusion of polycystic ovarian morphology (PCOM) from the diagnostic criteria for adolescent polycystic ovary syndrome (PCOS) has been proposed. We analyzed the profiles of adolescent women with suspected PCOS based on the Japan Society of Obstetrics and Gynecology (JSOG) diagnostic and Rotterdam criteria, excluding those with PCOM. METHODS: Thirteen- to twenty-one-year-old women with suspected or confirmed diagnosis of PCOS according to the JSOG and Rotterdam criteria were included in this study. Patient characteristics such as hormone levels and body mass index (BMI) were compared between the groups. Correlations between BMI and testosterone, and BMI and time to diagnosis were also analyzed. RESULTS: Twenty-nine patients were diagnosed with adolescent PCOS according to the JSOG criteria, and 11 patients according to the Rotterdam criteria after excluding the patients fulfilling the PCOM criteria. Serum testosterone levels were significantly higher in adolescents diagnosed with PCOS using the Rotterdam criteria than in those diagnosed using the JSOG criteria (p < 0.001). The obese group had significantly higher testosterone levels and a longer time from menarche to PCOS diagnosis. A positive correlation was observed between BMI and testosterone levels (r = 0.318, p = 0.014). CONCLUSION: Although adolescents with PCOS diagnosed using the Rotterdam criteria exhibited higher testosterone levels, which is a typical characteristic of this condition, the JSOG criteria may be useful for the early diagnosis of adolescent PCOS, including suspected cases. The differences between the two criteria may reflect the natural history of PCOS and its different reproductive and metabolic phenotypes.

Factors related to clearance of the small pelvic cavity during gynecologic laparoscopic surgery.

AIM: To identify factors influencing the Trendelenburg angle required during laparoscopic gynecological surgery. METHODS: Patients who underwent laparoscopic surgery at a single university hospital between May 1, 2019, and March 31, 2021 were enrolled. Data were extracted from the medical records, while magnetic resonance imaging scans and all laparoscopic surgery videos were retrospectively reviewed to assess the presence of the small intestine in the pelvic cavity as well as the adhesions at each site. Groups with and without the small intestine in the pelvic cavity, and those requiring a Trendelenburg angle above or below 13° were compared. RESULTS: In total, 219 patients were examined. The Trendelenburg angle was significantly higher (p = 0.004), while a significant increase in ovarian adhesions was observed (p = 0.033; odds ratio [OR], 2.30; 95% confidence interval [CI], 1.05-5.01) in the group without the presence of the small intestine in the pelvic cavity. Furthermore, the group requiring a Trendelenburg angle of ≥13° had significantly thicker subcutaneous fat (p = 0.044) and more ileal adhesions (p = 0.040, OR, 1.82; 95% CI, 1.03-3.23) than the group with an angle of <13°. CONCLUSION: Cases of ileal adhesions or thick subcutaneous fat are more likely to require a Trendelenburg angle of ≥13°. Therefore, Trendelenburg complications should be considered in this group. In addition, ovarian adhesions make it more difficult to exclude the small intestine from the small pelvic cavity, and may be associated with endometriosis.

Serum leucine-rich α2-glycoprotein as a possible marker for inflammatory status in endometriosis.

Purpose: This study aimed to investigate whether serum leucine-rich α2-glycoprotein (LRG) is a useful diagnostic biomarker for endometriosis, including the evaluation of treatment efficacy and exploration of LRG production in endometriotic lesions. Methods: Forty-three women with endometriomas were compared to 22 women with benign ovarian cysts and 30 women who underwent assisted reproduction as controls. Changes in serum LRG levels were assessed before and after surgery, and during dienogest treatment. LRG expression in endometriotic tissue samples was evaluated using immunoblotting. Results: Serum LRG levels in the endometrioma group (80.0 ± 36.3 µg/mL) were significantly higher than those in the benign ovarian cyst (65.1 ± 27.0 µg/mL, p = 0.0265) and control (57.8 ± 22.3 µg/mL, p = 0.0028) groups. Serum LRG levels after endometrioma surgery were significantly lower than preoperative levels (p = 0.0484). Serum LRG levels consistently decreased during dienogest treatment. LRG expression levels were significantly higher in endometriotic tissues than in the normal endometrium. Conclusion: Serum LRG, possibly derived from local and systemic origins, could be used as a potential biomarker for the diagnosis and treatment of endometriosis.

Promoter-specific dynamics of TATA-binding protein association with the human genome.

Transcription factor binding to target sites in vivo is a dynamic process that involves cycles of association and dissociation, with individual proteins differing in their binding dynamics. The dynamics at individual sites on a genomic scale have been investigated in yeast cells, but comparable experiments have not been done in multicellular eukaryotes. Here, we describe a tamoxifen-inducible, time-course ChIP-seq approach to measure transcription factor binding dynamics at target sites throughout the human genome. As observed in yeast cells, the TATA-binding protein (TBP) typically displays rapid turnover at RNA polymerase (Pol) II-transcribed promoters, slow turnover at Pol III promoters, and very slow turnover at the Pol I promoter. Turnover rates vary widely among Pol II promoters in a manner that does not correlate with the level of TBP occupancy. Human Pol II promoters with slow TBP dissociation preferentially contain a TATA consensus motif, support high transcriptional activity of downstream genes, and are linked with specific activators and chromatin remodelers. These properties of human promoters with slow TBP turnover differ from those of yeast promoters with slow turnover. These observations suggest that TBP binding dynamics differentially affect promoter function and gene expression, possibly at the level of transcriptional reinitiation/bursting.

Anti-Müllerian hormone levels in the diagnosis of adolescent polycystic ovarian syndrome: a systematic review and meta-analysis.

Polycystic ovary syndrome (PCOS) is an endocrine disorder that causes menstrual cycle irregularities and infertility. PCOS is diagnosed based on hyperandrogenism, polycystic ovarian morphology (PCOM), and an-/oligo-ovulation. Upregulation of anti-Müllerian hormone (AMH) in the serum of women with PCOS may be another suitable alternative diagnostic criterion for PCOM. However, previous meta-analyses have reported conflicting results due to the age-dependent decline in serum AMH levels. Therefore, we performed a meta-analysis to evaluate the threshold of AMH for the diagnosis of PCOS in adolescents and women in their early twenties. Fifteen trials were included in this meta-analysis. PCOS is diagnosed with either Rotterdam criteria, NIH, or AE-PCOS. AMH levels were significantly higher in adolescents with PCOS (weighted mean difference, 3.05; 95% confidence interval: 2.09-4.01) than in the control group. The cutoff values of AMH for the diagnosis of adolescent PCOS were 6.1, 6.26, 7.03, 7.11, 7.2, and 7.25 ng/mL in the studies that reported the usefulness of AMH levels. The summary receiver operating characteristic analysis of the diagnostic accuracy demonstrated that the specificity and sensitivity were 81% and 66.3%, respectively. Our meta-analysis demonstrates that AMH may be a useful diagnostic test for adolescent PCOS and, based on the previous studies included in the meta-analysis, its cutoff value was estimated to be 6-7 ng/mL.

Effect of salpingectomy on ovarian reserve: A systematic review and meta-analysis.

AIM: To determine the effect of salpingectomy on ovarian reserve. METHODS: PubMed, EMBASE, Web of Science, Dynamed plus, and Cochrane Controlled Trials Register databases were searched from their inception to December 2020 to identify relevant studies, including cross-sectional studies, retrospective studies, and randomized controlled trials. Studies that compared anti-Müllerian hormone (AMH) levels and/or antral follicle count (AFC) between the control and salpingectomy groups or before and after surgery were included. RESULTS: Twenty-one articles were included in the systematic review. Meta-analyses were performed on 16 studies in which data were presented as mean ± SD values. A meta-analysis comparing AMH levels before and after surgery in the same patients showed no significant decrease in all cases, irrespective of whether it was unilateral or bilateral salpingectomy. There was no significant decrease in the AFC in the meta-analysis comparing levels before and after bilateral salpingectomy, either. In contrast, in the case-controlled study the salpingectomy group had significantly lower levels of AMH in all meta-analyses of unilateral and bilateral surgery (mean difference: -0.31, 95% confidence interval [CI]: -0.55, -0.07), only unilateral cases (mean difference: -0.28, 95% CI: -0.50, -0.06), and only bilateral cases (mean difference: -0.71, 95% CI: -1.19, -0.23). The salpingectomy group that included unilateral and bilateral cases had significantly lower AFC compared with no-surgery controls (mean difference: -1.31, 95% CI: -2.13, -0.48). CONCLUSION: Although not conclusive, it does appear that patients who underwent salpingectomy (either unilateral or bilateral) have a decreased ovarian reserve.

Effect of hypothyroidism and thyroid autoimmunity on the ovarian reserve: A systematic review and meta-analysis.

BACKGROUND: Evidence suggests that hypothyroidism and thyroid autoimmunity (TAI) are possibly associated with ovarian dysfunction. This meta-analysis aimed to investigate whether hypothyroidism and/or TAI affect the ovarian reserve and evaluated using the anti-Mullerian hormone (AMH). METHODS: PubMed, EMBASE, Web of Science, and Cochrane Controlled Trials Register databases from inception to October 2020 were searched to identify relevant studies. Studies comparing the AMH levels between the control and the affected groups were included in the data synthesis. The primary endpoint in the meta-analysis was AMH levels compared with the controls. MAIN FINDINGS: Nine trials were included in the analysis. The AMH levels were significantly lower in the adults with euthyroid TAI (mean difference -0.12, [95% CI: -0.18 to -0.06]). The AMH levels tended to be lower in subclinical hypothyroidism and overt hypothyroidism than in the control group, although the differences were not significant. The AMH levels were significantly higher in the euthyroid TAI group in the adolescents (mean difference 2.51, [95% CI 1.82 to 3.21]). CONCLUSION: TAI and hypothyroidism may affect the ovarian reserve. The opposite effects on AMH levels depending on age suggest that TAI may be implicated in the depletion of follicles in adults following extensive activation of primordial follicles in adolescence.

Spatial organization of a model 15-member human gut microbiota established in gnotobiotic mice.

Knowledge of the spatial organization of the gut microbiota is important for understanding the physical and molecular interactions among its members. These interactions are thought to influence microbial succession, community stability, syntrophic relationships, and resiliency in the face of perturbations. The complexity and dynamism of the gut microbiota pose considerable challenges for quantitative analysis of its spatial organization. Here, we illustrate an approach for addressing this challenge, using (i) a model, defined 15-member consortium of phylogenetically diverse, sequenced human gut bacterial strains introduced into adult gnotobiotic mice fed a polysaccharide-rich diet, and (ii) in situ hybridization and spectral imaging analysis methods that allow simultaneous detection of multiple bacterial strains at multiple spatial scales. Differences in the binding affinities of strains for substrates such as mucus or food particles, combined with more rapid replication in a preferred microhabitat, could, in principle, lead to localized clonally expanded aggregates composed of one or a few taxa. However, our results reveal a colonic community that is mixed at micrometer scales, with distinct spatial distributions of some taxa relative to one another, notably at the border between the mucosa and the lumen. Our data suggest that lumen and mucosa in the proximal colon should be conceptualized not as stratified compartments but as components of an incompletely mixed bioreactor. Employing the experimental approaches described should allow direct tests of whether and how specified host and microbial factors influence the nature and functional contributions of "microscale" mixing to the dynamic operations of the microbiota in health and disease.

A histopathological analysis of spontaneous neoplastic and non-neoplastic lesions in aged male RccHan:WIST rats.

Histopathological information about spontaneous lesions in aged Hannover Wistar rats is limited. In this study, we describe spontaneous lesions found in 39 male RccHan:WIST rats used as a control in a carcinogenicity study. Neoplastic lesions were frequently seen in the endocrine system, such as pituitary adenomas in the pars distalis. This strain exhibited a high incidence of thymoma (10.3%), compared to other strains. We encountered an oligodendroglioma, a pituitary adenoma of the pars intermedia, and a prostate adenocarcinoma, which are comparatively rare in rats. While the variety and incidence of non-neoplastic lesions were similar to those in other strains, several interesting lesions occurred with relatively high incidence, including "harderianization" of the extraorbital lacrimal gland, common bile duct ectasia, and hyperplasia of pulmonary endocrine cells in the lung. Furthermore, comparative analyses demonstrated that the severity of chronic progressive nephropathy and murine progressive cardiomyopathy in RccHan:WIST rats was less than that in F344 rats.

Xist Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome.

To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.

Formation of nuclear bodies by the lncRNA Gomafu-associating proteins Celf3 and SF1.

Gomafu/MIAT/Rncr2 is a long noncoding RNA that has been proposed to control retinal cell specification, stem cell differentiation and alternative splicing of schizophrenia-related genes. However, how Gomafu controls these biological processes at the molecular level has remained largely unknown. In this study, we identified the RNA-binding protein Celf3 as a novel Gomafu-associating protein. Knockdown of Celf3 led to the down-regulation of Gomafu, and cross-link RNA precipitation analysis confirmed specific binding between Celf3 and Gomafu. In the neuroblastoma cell line Neuro2A, Celf3 formed novel nuclear bodies (named CS bodies) that colocalized with SF1, another Gomafu-binding protein. Gomafu, however, was not enriched in the CS bodies; instead, it formed distinct nuclear bodies in separate regions in the nucleus. These observations suggest that Gomafu indirectly modulates the function of the splicing factors SF1 and Celf3 by sequestering these proteins into separate nuclear bodies.

Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging.

Microbes in nature frequently function as members of complex multitaxon communities, but the structural organization of these communities at the micrometer level is poorly understood because of limitations in labeling and imaging technology. We report here a combinatorial labeling strategy coupled with spectral image acquisition and analysis that greatly expands the number of fluorescent signatures distinguishable in a single image. As an imaging proof of principle, we first demonstrated visualization of Escherichia coli labeled by fluorescence in situ hybridization (FISH) with 28 different binary combinations of eight fluorophores. As a biological proof of principle, we then applied this Combinatorial Labeling and Spectral Imaging FISH (CLASI-FISH) strategy using genus- and family-specific probes to visualize simultaneously and differentiate 15 different phylotypes in an artificial mixture of laboratory-grown microbes. We then illustrated the utility of our method for the structural analysis of a natural microbial community, namely, human dental plaque, a microbial biofilm. We demonstrate that 15 taxa in the plaque community can be imaged simultaneously and analyzed and that this community was dominated by early colonizers, including species of Streptococcus, Prevotella, Actinomyces, and Veillonella. Proximity analysis was used to determine the frequency of inter- and intrataxon cell-to-cell associations which revealed statistically significant intertaxon pairings. Cells of the genera Prevotella and Actinomyces showed the most interspecies associations, suggesting a central role for these genera in establishing and maintaining biofilm complexity. The results provide an initial systems-level structural analysis of biofilm organization.

Peripheral serotonergic neurons regulate gut motility and anxiety-like behavior.

Serotonergic circuits in the central nervous system play important roles in regulating mood and behavior, yet the functions of peripheral serotonergic neurons are less understood. Here, we engineered mice lacking the serotonin-producing enzyme Tph2 in peripheral neurons but with intact Tph2 in central neurons. In contrast to mice lacking Tph2 in all neurons, mice lacking Tph2 in peripheral serotonergic neurons did not exhibit increased territorial aggression. However, similar to the total body Tph2 knockout (KO) mice, the conditional KO animals exhibited reduced gut motility and decreased anxiety-like behavior. These observations reveal that peripheral serotonergic neurons contribute to control of intestinal motility and anxiety-like behavior and suggest that therapeutics targeting this subset of peripheral neurons could be beneficial.

Recurrent Bouts of Fever and Transient Hydrosalpinx Manifested in a Female Carrying MEFV G304R Gene Variant: A Case Report.

Familial Mediterranean fever (FMF) is an inherited autoinflammatory disease characterized by recurrent bouts of fever and serositis. Mediterranean Fever (MEFV) gene mutations may cause not just FMF but various serositis including arthritis, enterocolitis, aseptic meningitis, pulmonary disease, and pericarditis. In this report, we present a 44-year-old female carrying MEFV gene variant. She was admitted to our hospital with a high fever, right back pain during inspiration, and lower-left abdominal pain. Laboratory findings showed high inflammatory response. Computed tomography (CT) indicated pleurisy of the right lobe and inflammation of the left uterine appendage. Transvaginal sonography and magnetic resonance imaging (MRI) indicated hydrosalpinx of the left oviduct. The symptoms of recurrent fever and transient serositis suggested FMF, and abdominal pain was resolved after taking colchicine. Later, it turned out that she had MEFV gene mutation (exon2 G304R heterozygous). Although she did not meet the criteria of FMF, this is the first reported MEFV variant carrier with transient hydrosalpinx. Attacks in female patients with FMF are triggered by menstruation. Moreover, FMF and associated amyloidosis may cause both male and female infertility. Although male patients with FMF may present with acute scrotum, diagnostic criteria of FMF do not include inflammation of uterine appendages. Internal medicine physicians need to cooperate with gynecologists to diagnose female patients carrying MEFV gene variants.

Effects of epidural analgesia on labor length, instrumental delivery, and neonatal short-term outcome.

PURPOSE: We aimed to clarify whether the short-term adverse neonatal outcomes associated with epidural analgesia are due to the epidural analgesia itself or to the instrumental delivery. METHODS: A retrospective case-control study was conducted to evaluate the relationship between epidural analgesia, labor length, and perinatal outcomes. A total of 350 pregnant women at term who delivered under epidural analgesia (cases) were compared with 1400 patients without epidural analgesia (controls). RESULTS: Vacuum extraction (6.5 vs. 2.9 %) and cesarean section (19.9 vs. 11.1 %) were more frequently performed in the cases than controls (p < 0.001). Using a Kaplan-Meier algorithm, it was determined that the mean lengths of the 1st and 2nd stages of labor and the overall durations of labor and delivery were significantly longer in cases compared with controls. A Cox regression analysis showed that the longer labor remained even after adjustment for parity. The neonatal variables stratified by mode of delivery were not different in cases and controls, except for a slightly lower umbilical arterial pH in spontaneous delivery for the cases group. However, the Apgar scores and umbilical arterial pH were significantly lower in the neonates delivered by vacuum extraction compared with those in the neonates delivered by spontaneous delivery or cesarean section, regardless of whether epidural analgesia was performed. A multivariable analysis showed that vacuum extraction much more consistently affected the arterial pH than the analgesia itself (the ß coefficients were -0.036 for epidural analgesia vs. -0.050 for vacuum extraction). CONCLUSION: Epidural analgesia was associated with slowly progressing labor, thus resulting in an increased rate of instrumental delivery. This instrumental delivery appears to adversely affect the neonatal outcomes more strongly than the analgesia itself.

Spleen-specific development of germinal centers in rats treated with antithyroid drugs.

The antithyroid drugs (ATDs) methimazole (MMI) and propylthiouracil (PTU) have been used for treatment of hyperthyroidism for more than several decades, despite the fact that they are associated with adverse drug reactions that are thought to be autoimmune mediated. We therefore examined histopathologic responses in the immune system in male and female rats given MMI (2, 20 and 200 mg/kg/day, p.o., in experiment 1; 200 mg/kg/day, p.o., in experiment 3) or PTU (25 and 250 mg/kg/day, p.o., in experiment 2; 200 mg/kg/day, p.o., in experiment 3) for two weeks. In experiments 1 and 2, highest doses of MMI and PTU induced histopathologic changes in the spleen consistent with those in experiment 3 without any changes in the other peripheral lymphoid organs and tissues. In experiment 3, histopathological evaluation of the spleen along with hematological and bone marrow examinations were performed. In both male and female rats, MMI or PTU induced histopathological changes in the spleen characterized by development of germinal centers and an increase in the number of IgG-positive plasma cells in the red pulp; these changes were most prevalent in the MMI-treated female rats. Total red and white blood cell counts were decreased in the MMI-treated male and female rats; lymphocytes and monocytes were lower in male and female rats, respectively. Bone marrow nucleated cells were significantly lower in the MMI-treated males. This is the first study to demonstrate that ATDs induce spleen specific B-cell reactions in rats.