Javier Regadera

A murine model of falciparum-malaria by in vivo selection of competent strains in non-myelodepleted mice engrafted with human erythrocytes.

To counter the global threat caused by Plasmodium falciparum malaria, new drugs and vaccines are urgently needed. However, there are no practical animal models because P. falciparum infects human erythrocytes almost exclusively. Here we describe a reliable falciparum murine model of malaria by generating strains of P. falciparum in vivo that can infect immunodeficient mice engrafted with human erythrocytes. We infected NODscid/β2m−/− mice engrafted with human erythrocytes with P. falciparum obtained from in vitro cultures. After apparent clearance, we obtained isolates of P. falciparum able to grow in peripheral blood of engrafted NODscid/β2m−/− mice. Of the isolates obtained, we expanded in vivo and established the isolate Pf3D70087/N9 as a reference strain for model development. Pf3D70087/N9 caused productive persistent infections in 100% of engrafted mice infected intravenously. The infection caused a relative anemia due to selective elimination of human erythrocytes by a mechanism dependent on parasite density in peripheral blood. Using this model, we implemented and validated a reproducible assay of antimalarial activity useful for drug discovery. Thus, our results demonstrate that P. falciparum contains clones able to grow reproducibly in mice engrafted with human erythrocytes without the use of myeloablative methods.

Thyroid Hormone Receptor β1 Acts as a Potent Suppressor of Tumor Invasiveness and Metastasis

Loss of thyroid hormone receptors (TR) is a common feature in some tumors, although their role in tumor progression is currently unknown. We show here that expression of TRbeta1 in hepatocarcinoma and breast cancer cells reduces tumor growth, causes partial mesenchymal-to-epithelial cell transition, and has a striking inhibitory effect on invasiveness, extravasation, and metastasis formation in mice. In cultured cells, TRbeta1 abolishes anchorage-independent growth and migration, blocks responses to epidermal growth factor, insulin-like growth factor-I, and transforming growth factor beta, and regulates expression of genes that play a key role in tumorigenicity and metastatic growth. The receptor disrupts the mitogenic action of growth factors by suppressing activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling pathways that are crucial for cell proliferation and invasiveness. Furthermore, increased aggressiveness of skin tumors is found in genetically modified mice lacking TRs, further demonstrating the role of these receptors as inhibitors of tumor progression. These results define a novel role for the thyroid hormone receptor as a metastasis suppressor gene, providing a starting point for the development of novel therapeutic strategies for the treatment of human cancer.

A quantitative morphological study of human Leydig cells from birth to adulthood

SummaryHuman testicular specimens were obtained from biopsies and autopsies covering the period from birth to adulthood. The number of testosterone-containing Leydig cells was determined using the peroxidase-anti-peroxidase method. This number decreased markedly from 3–6 months of age to the end of the first year of life and, up to 6 years of age, only a small number of testosterone-containing cells was found. From 6 years onwards the number of Leydig cells progressively increased. Ultrastructural examination revealed four types of Leydig cells: (1) fetal-type Leydig cells (from birth to 1 year of age) with round nuclei, abundant smooth endoplasmic reticulum and mitochondria with tubular cristae; (2) infantile-type Leydig cells (from birth to 8–10 years of age), showing a multilobated nucleus, moderately abundant smooth endoplasmic reticulum, some lipid droplets and mitochondria with parallel cristae; (3) prepubertal, partially differentiated Leydig cells (from 6 years of age onwards) with regularly-outlined round nuclei, abundant smooth endoplasmic reticulum, mitochondria with tubular cristae, and some lipid droplets and lipofuscin granules; and (4) mature adult Leydig cells (from 8–10 years of age onwards). The ultrastructure of the infantile-type Leydig cells and the lack of delay between the disappearance of the fetal-type Leydig cells and the appearance of infantile-type Leydig cells suggest that fetal-type Leydig cells give rise to the infantile-type Leydig cells. Before puberty, myofibroblast-like precursor cells differentiate into the prepubertal, partially differentiated Leydig cells, which complete their differentiation into the adult Leydig cells.

Hypothyroidism Enhances Tumor Invasiveness and Metastasis Development

Background Whereas there is increasing evidence that loss of expression and/or function of the thyroid hormone receptors (TRs) could result in a selective advantage for tumor development, the relationship between thyroid hormone levels and human cancer is a controversial issue. It has been reported that hypothyroidism might be a possible risk factor for liver and breast cancer in humans, but a lower incidence of breast carcinoma has been also reported in hypothyroid patients Methodology/Principal Findings In this work we have analyzed the influence of hypothyroidism on tumor progression and metastasis development using xenografts of parental and TRβ1–expressing human hepatocarcinoma (SK-hep1) and breast cancer cells (MDA-MB-468). In agreement with our previous observations tumor invasiveness and metastasis formation was strongly repressed when TRβ–expressing cells were injected into euthyroid nude mice. Whereas tumor growth was retarded when cells were inoculated into hypothyroid hosts, tumors had a more mesenchymal phenotype, were more invasive and metastatic growth was enhanced. Increased aggressiveness and tumor growth retardation was also observed with parental cells that do not express TRs. Conclusions/Significance These results show that changes in the stromal cells secondary to host hypothyroidism can modulate tumor progression and metastatic growth independently of the presence of TRs on the tumor cells. On the other hand, the finding that hypothyroidism can affect differentially tumor growth and invasiveness can contribute to the explanation of the confounding reports on the influence of thyroidal status in human cancer.

The apical mitochondria-rich cells of the mammalian epididymis.

Summary The morphology and function of the apical mitochondria‐rich cells in the mammalian ductus epididymidis epithelium are revised. These cells are similar in all mammalian species studied. Apical mitochondria‐rich cells are scarce (1–5 cells/100 principal cells) and are mainly found in the initial epididymal segments. Their morphology varies from slender cells that extend from the basal lamina to the epididymal lumen, to round cells that protrude into the lumen and are not in contact with the basal lamina. Their cytoplasm is more electron‐dense than that of principal cells and contains more mitochondria which, in some species, are surrounded by rough endoplasmic reticulum cisternae. The adluminal cytoplasm displays a few short microvilli and contains many acid phosphatase positive vesicles. Apical mitochondria‐rich cells differ from the principal cells in some histochemical features such as: (a) different lectin‐staining pattern; (b) more intense reaction to the enzymatic activities: carbonic anhydrase, Ca2+‐ATPase, peanut‐agglutinin‐sialidase, NADP dehydrogenase, succinate dehydrogenase, α‐galactosidase and β‐galactosidase; (c) more intense immunoreaction to several cytokeratin types and to estradiol‐related receptor protein; (d) weaker immunoreaction to epithelial membrane antigen and to retinol‐binding protein. Although the function of the apical mitochondria‐rich cells is still unknown, the following possible functions have been suggested: holocrine secretion; cooperation with the principal cells in epididymal reabsorption of testicular fluid; and acidification of epididymal fluid. Experimental results suggest that differentiation and maintenance of apical mitochondria‐rich cells are not under androgen control and that these cells are sensitive to estrogen stimulation.

Antifungal Efficacy of GM237354, a Sordarin Derivative, in Experimental Oral Candidiasis in Immunosuppressed Rats

ABSTRACT GM237354 is a novel sordarin derivative with a broad spectrum of potent activity against a wide range of fungi. The members of this new class of antifungal agents act as potent inhibitors of fungal protein synthesis. In this study, the therapeutic effects of GM237354 were investigated in a novel experimental oral Candida albicansinfection model in immunosuppressed rats. The animals were immunosuppressed with dexamethasone in their drinking water and infected on three alternate days. GM237354 was given three times per day for seven consecutive days at 1.25, 2.5, 5, or 10 mg/kg of body weight per dose. In addition, to provide a preliminary idea of the correlation between regimen administration and therapeutic efficacy, GM237354 was administered to two additional groups of rats at 5 mg/kg once or twice a day for 7 days. The drug efficacy was assessed microbiologically, histologically, and by a morphometric study of lesions. Evident agreement was observed among results obtained by the different methods in all of the animals studied. Microbiologically, the efficacy of GM237354 was determined by measuring the number of C. albicans organisms in the oral cavities of rats in the middle (day 4) and at the end (day 7) of the treatment. GM237354 administered at 5, 7.5, 10, 15, or 30 mg/kg/day for 7 days significantly reduced the number of CFU in the oral cavities of treated rats compared with the number of CFU in the oral cavities of the untreated controls. A significant reduction was also observed when GM237354 was administered at 7.5, 10, 15, or 30 mg/kg/day for 4 days. Furthermore, C. albicans was not detected in oral swabs from any infected rats after 1 week of treatment when GM237354 was administered at 15 or 30 mg/kg/day or after 4 days of treatment at 30 mg/kg/day. Histologically, untreated control animals showed extensive colonization of the epithelium of the dorsal tongue by numerous hyphae. Animals treated with GM237354 at 7.5 mg/kg/day showed small areas with superficial hyphal penetration into the epithelium that produced intraepithelial microabscesses. However, animals treated with GM237354 at 15 mg/kg/day showed multiple regenerative areas of the covering epithelium, and only focalized zones of the tongue surface were occupied by hyphae. No hyphal colonization of the epithelium was seen in rats treated with GM237354 at 30 mg/kg/day and which showed extensive areas of epithelial regeneration of the tongue. The histopathology findings were confirmed by morphometry studies, and the percentage of epithelium occupied byC. albicans hyphae decreased from 17.5% in the control group to 4.8 and 0.1% in animals treated with GM237354 at 7.5 and 15 mg/kg/day, respectively. These results demonstrated that the sordarin derivative GM237354 was effective against experimental oral candidiasis in immunosuppressed rats, and further studies are needed to determine the potential of GM237354 for use in the treatment of this infection in humans.

Sterile Inflammation in Acetaminophen-induced Liver Injury Is Mediated by Cot/tpl2

Background: MAP3K8 (Cot/tpl2) activates MKK1/2-Erk1/2 upon stimulation of receptors from the Toll-like/interleukin-1 receptor superfamily. Results: Cot/tpl2 plays an essential role in acetaminophen-induced liver injury by modulating the generation of inflammatory signals induced by necrotic cells. Conclusion: Sterile inflammatory processes triggered by tissue damage are modulated by Cot/tpl2. Significance: Cot/tpl2 contributes to the development of pathologies associated with inflammation triggered by damage-associated molecular patterns. Cot/tpl2 (MAP3K8) activates MKK1/2-Erk1/2 following stimulation of the Toll-like/IL-1 receptor superfamily. Here, we investigated the role of Cot/tpl2 in sterile inflammation and drug-induced liver toxicity. Cot/tpl2 KO mice exhibited reduced hepatic injury after acetaminophen challenge, as evidenced by decreased serum levels of both alanine and aspartate aminotransferases, decreased hepatic necrosis, and increased survival relative to Wt mice. Serum levels of both alanine and aspartate aminotransferases were also lower after intraperitoneal injection of acetaminophen in mice expressing an inactive form of Cot/tpl2 compared with Wt mice, suggesting that Cot/tpl2 activity contributes to acetaminophen-induced liver injury. Furthermore, Cot/tpl2 deficiency reduced neutrophil and macrophage infiltration in the liver of mice treated with acetaminophen, as well as their hepatic and systemic levels of IL-1α. Intraperitoneal injection of damage-associated molecular patterns from necrotic hepatocytes also impaired the recruitment of leukocytes and decreased the levels of several cytokines in the peritoneal cavity in Cot/tpl2 KO mice compared with Wt counterparts. Moreover, similar activation profiles of intracellular pathways were observed in Wt macrophages stimulated with Wt or Cot/tpl2 KO damage-associated molecular patterns. However, upon stimulation with damage-associated molecular patterns, the activation of Erk1/2 and JNK was deficient in Cot/tpl2 KO macrophages compared with their Wt counterparts; an effect accompanied by weaker release of several cytokines, including IL-1α, an important component in the development of sterile inflammation. Taken together, these findings indicate that Cot/tpl2 contributes to acetaminophen-induced liver injury, providing some insight into the underlying molecular mechanisms.

Xanthogranulomatous Funiculitis and Orchiepididymitis: Report of 2 Cases With Immunohistochemical Study and Literature Review

Two patients with xanthogranulomatous inflammation are described, one with involvement of the spermatic cord and the other with 1 testicle and epididymis affected. To our knowledge, only 12 cases of xanthogranulomatous orchiepididymitis have been reported previously, one of which also presented a xanthogranulomatous funiculitis. Clinically, our patients presented with spermatic cord enlargement (case 1) and chronic orchitis that did not respond to treatment with antibiotics (case 2). Histopathologically, both cases showed extensive xanthogranulomatous inflammation with numerous foamy macrophages that were associated with colonies of microorganisms suggestive of actinomyces in case 1. Additionally, Escherichia coli was cultured from the surgical specimen of case 2. The possible underlying pathology may be diabetes in case 1 and phlebitis associated with chronic orchitis in case 2. Differential diagnoses with other lesions that are rich in macrophages, such as malakoplakia, and those testicular neoplasms without serologic tumor markers are discussed.

Androgen Insensitivity Syndrome An Immunohistochemical, Ultrastructural, and Morphometric Study

OBJECTIVE To evaluate the morphometric, immunohistochemical, and ultrastructural lesions of the testes in prepubertal and adult patients with androgen insensitivity syndrome. METHODS We examined the testicular biopsy using immunohistochemistry for vimentin, smooth muscle actin, and collagen IV antigens. Quantification of seminiferous tubules and testicular interstitium was performed in prepubertal and adult patients with androgen insensitivity syndrome and results were compared with normal testes from both infants and adults. RESULTS The adult testes presented nodular and diffuse lesions that consisted of Sertoli-cell-only seminiferous tubules. Two types of Sertoli cells could be distinguished, namely, immature vimentin-positive Sertoli cells and nearly mature Sertoli cells. In the nodules, the lamina propria was thin and contained a scant number of actin-positive peritubular cells. Leydig cells were hyperplastic. The prepubertal patients showed only diffuse lesions characterized by Sertoli cell hyperplasia, decreased germ cell numbers, and a discontinuous immunoreaction to collagen IV. CONCLUSIONS The testicular lesions in androgen insensitivity syndrome are probably caused by primary alterations that begin during gestation. These lesions become progressively more pronounced at puberty, when the nodular lesion pattern (adenomas) is completely developed.

Histological, Histochemical and Ultrastructural Variations along the Length of the Human vas deferens before and after Puberty

Vasa deferentia of 20 human adult males and 8 children were divided into four portions (proximal, middle, distal and terminal) for histological, histochemical and ultrastructural studies. The epithelium of the adult vas deferens was formed of columnar and basal cells. Three types of columnar cells were observed: (1) principal cells with PAS-positive cytoplasmic granules (lysosomes); (2) dark or pencil cells with a dark cytoplasm and nucleoplasm and cytoplasmic lipid vacuoles, and (3) mitochondrion-rich cells with numerous mitochondria similar to those of principal cells. All these cell types showed stereocilia and irregular convoluted nuclei, some of them containing granular, PAS-positive intranuclear inclusions. Principal cells were the most frequent cell type in the proximal portion but their number gradually decreased along the length of the vas deferens, while the proportion of both pencil cells and mitochondrion-rich cells increased. The muscular coat comprised three muscular layers: (1) inner longitudinal, (2) middle oblique (circular in the distal and terminal portions), and (3) outer longitudinal. The functional significance of regional variations is discussed. The infant vas deferens showed a columnar cell type only. However, cells either with numerous lysosomes or with numerous mitochondria were occasionally found. No regional variations were observed in the infant vas.