Divergent macrophage responses to Mycobacterium bovis among naturally exposed uninfected and infected cattle.

Mycobacterium bovis, the causative agent of bovine tuberculosis (TB), is a successful pathogen that remains an important global threat to livestock. Cattle naturally exposed to M. bovis normally become reactive to the M. bovis-purified protein derivative (tuberculin) skin test; however, some individuals remain negative, suggesting that they may be resistant to infection. To better understand host innate resistance to infection, 26 cattle from herds with a long history of high TB prevalence were included in this study. We investigated the bactericidal activity, the production of reactive oxygen and nitrogen species and the TB-related gene expression profile after in vitro M. bovis challenge of monocyte-derived macrophages from cattle with TB (n=17) and from non-infected, exposed cattle (in-contacts, n=9). The disease status was established based on the tuberculin skin test and blood interferon-gamma test responses, the presence of visible lesions at inspection on abattoirs and the histopathology and culture of M. bovis. Although macrophages from TB-infected cattle enabled M. bovis replication, macrophages from healthy, exposed cattle had twofold lower bacterial loads, overproduced nitric oxide and had lower interleukin (IL)-10 gene expression (P⩽0.05). Higher mRNA expression levels of inducible nitric oxide synthase, C-C motif chemokine ligand 2 and IL-12 were observed in macrophages from all in-contact cattle than in macrophages from their TB-infected counterparts, which expressed more tumour necrosis factor-α; however, the differences were not statistically significant owing to individual variation. These results confirm that macrophage bactericidal responses have a crucial role in innate resistance to M. bovis infection in cattle.

Differential expression of solute carrier family 11a member 1 and inducible nitric oxide synthase 2 in skin biopsies from leprosy patients.

BACKGROUND: Leprosy is a chronic granulomatous infection caused by Mycobacterium leprae, an intracellular parasite that resides within macrophages and cannot be eliminated effectively. Solute carrier family 11a member 1 (Slc11a1) and inducible nitric oxide synthase (iNOS), both expressed in macrophages, play major roles in host defense against several intracellular pathogens. However, the roles of these molecules in natural infection with M. leprae remain unknown. OBJECTIVE: We aimed to investigate the expression of Slc11a1 and iNOS in macrophages (CD68+ cells) infiltrating skin lesions in leprosy. METHODS: Skin biopsies from 48 Mexican patients of leprosy [(33 lepromatous (LL), 15 tuberculoid (TT)] and from 10 healthy controls, were subjected to immunohistochemistry to determine expression of CD68, Slc11a1 and iNOS. RESULTS: We found a high expression of Slc11a1 and iNOS in most lepromatous leprosy samples. In tuberculoid leprosy samples, Slc11a1 expression was moderate or low, and that of iNOS was almost always low. In addition, Slc11a1 and iNOS expression levels were positively associated with bacillary loads in lepromatous leprosy lesions (P=0.05). CONCLUSIONS: These observations suggest that M. leprae infection promotes the expression of Slc11a1 and iNOS in macrophages and that lepromatous leprosy can occur despite this response.

An approach to the immunophenotypic features of circulating CD4⁺NKG2D⁺ T cells in invasive cervical carcinoma.

BACKGROUND: NKG2D, an activating immunoreceptor, is primarily restricted to NK cells and CD8(+) T cells. The existence of an atypical cytotoxic CD4(+)NKG2D(+) T cell population has also been found in patients with autoimmune dysfunctions. Nonetheless, contradictory evidence has categorized this population with a regulatory rather than cytotoxic role in other situations. These confounding data have led to the proposal that two distinct CD4(+)NKG2D(+) T cell subsets might exist. The immune response elicited in cervical cancer has been characterized by apparent contradictions concerning the role that T cells, in particular T-helper cells, might be playing in the control of the tumor growth. Interestingly, we recently reported a substantial increase in the frequency of CD4(+)NKG2D(+) T cells in patients with cervical intraepithelial neoplasia grade-1. However, whether this particular population is also found in patients with more advanced cervical lesions or whether they express a distinctive phenotype remains still to be clarified. In this urgent study, we focused our attention on the immunophenotypic characterization of CD4(+)NKG2D(+) T cells in patients with well-established cervical carcinoma and revealed the existence of at least two separate CD4(+)NKG2D(+) T cell subsets defined by the co-expression or absence of CD28. RESULTS: Patients with diagnosis of invasive cervical carcinoma were enrolled in the study. A group of healthy individuals was also included. Multicolor flow cytometry was used for exploration of TCR alpha/beta, CD28, CD158b, CD45RO, HLA-DR, CD161, and CD107a. A Luminex-based cytokine kit was used to quantify the levels of pro- and anti-inflammatory cytokines. We found an increased percentage of CD4(+)NKG2D(+) T cells in patients with cervical cancer when compared with controls. Accordingly with an increase of CD4(+)NKG2D(+) T cells, we found decreased CD28 expression. The activating or degranulation markers HLA-DR, CD161, and CD107a were heterogeneously expressed. The levels of IL-1beta, IL-2, TNF-alpha, and IL-10 were negatively correlated with the percentages of CD4(+)NKG2D(+) T cells in patients with cervical carcinoma. CONCLUSIONS: Taken together, our results reveal the existence of two separate CD4(+)NKG2D(+) T cell subsets defined by the co-expression or absence of CD28, the latter more likely to be present in patients with cervical cancer.

STAT3 activation is required for the antiapoptotic effects of prolactin in cervical cancer cells.

BACKGROUND: Prolactin (PRL) has been implicated in the development of different types of cancer. However, signaling pathways might be activated depending on various forms of prolactin receptor (PRLR). JAK/STAT is an important pathway associated with PRL effects. The activation of JAK/STAT pathway might activate antiapoptotic genes that could importantly lead to progression of tumorigenesis. Recently, we have reported that PRL is associated with cell survival by inhibition of apoptosis and the precise activated signaling pathways for this process are still questioned. The purpose of this study was to evaluate the activation of different signaling pathways in response to PRL as well as to identify the induction of antiapoptotic genes. METHODS: Cervical cancer cell lines HeLa, SiHa and C-33 A were stimulated with PRL (200 ng/mL) for 30 and 60 min and non stimulated cells were used to measure basal protein expression. Inhibition assays were performed by using Jak2 specific inhibitor AG490, either alone or in combination with PRL for 48 h. Western blot were carried out to evaluate protein induction of the different signaling pathways and antiapoptotic proteins. Significant effects were determined by using ANOVA test. RESULTS: STAT3 was significantly activated in cervical cancer lines in comparison with non-tumorigenic keratinocytes HaCaT. No significant differences were found when analyzing MAPK and PI3K signaling pathways. An increase of antiapoptotic genes Bcl-xl, Bcl-2, survivin and Mcl-1 was observed after stimulus with PRL; however, after inhibition with AG490, the induction of antiapoptotic genes was decreased. CONCLUSION: Our data suggests that STAT3 is an important signaling pathway activated by PRL in cervical cancer cells and it modulates the induction of antiapoptotic genes. Blocking STAT3 could represent a possible therapeutic strategy in cervical cancer.

Expression of autocrine prolactin and the short isoform of prolactin receptor are associated with inflammatory response and apoptosis in monocytes stimulated with Mycobacterium bovis proteins.

Increased levels of prolactin (PRL) have recently been associated with carcinogenesis and the exacerbation of autoimmune diseases, and might be involved in the progression of tuberculosis (TB). To investigate the relationship between PRL and prolactin receptor (PRLr) expression with inflammatory response and apoptosis in monocytes, we used THP-1 cells stimulated with antigens of the Mycobacterium bovis AN5 strain culture filtrate protein (CFP-M. bovis). Western blot (WB), real-time Polymerase chain reaction (PCR), and immunocytochemistry were performed to identify both PRL and PRLr molecules. PRL bioactivity and proinflammatory cytokine detection were assessed. The results showed that PRL and PRLr messenger RNA (mRNA) were synthesized in THP-1 monocytes induced with CFP-M. bovis at peaks of 176- and 404-fold, respectively. PRL forms of 60 and 80kDa and PRLr isoforms of 40, 50, and 65kDa were also identified as time-dependent, while 60-kDa PRL, as well as 40-, and 50-kDa PRLr, were found as soluble forms in culture media and later in the nucleus of THP-1 monocytes. PRL of 60kDa released by monocytes exhibited bioactivity in Nb2 cells, and both synthesized PRL and synthesized PRLr were related with nitrite and proinflammatory cytokine levels proapoptotic activity in CFP-M. bovis-induced monocytes. Our results suggest the overexpression of a full-autocrine loop of PRL and PRLr in monocytes that enhances the inflammatory response and apoptosis after priming with M. bovis antigens.

Prolactin and prolactin receptor expression in cervical intraepithelial neoplasia and cancer.

Prolactin receptor (PRLR) overexpression could play a role in tumorigenesis. The aim of this study was to determine prolactin (PRL) and PRLR expression in biopsies from patients with precursor lesions and uterine cervical cancer. PRLR expression was analyzed in 63 paraffin-embedded biopsies of uterine cervical tissue. In total, eleven low-grade squamous intraepithelial lesions (LSIL), 23 high-grade squamous intraepithelial lesions (HSIL), 21 uterine cervical cancers (UCC) and 8 normal epithelium (NE) were examined using immunoperoxidase staining and Western blot analysis. Additionally, PRL expression was identified in human cervical cancer serum and tissues. The PRLR expression was found to be significantly increased in cervical cancer in comparison with normal tissue and precursor lesions (P < 0.0003). The presence of the long isoform of the PRLR was observed only in cervical cancer tissues. Serum PRL levels were normal in all samples and local prolactin expression was similar in precursor lesions and cervical cancer by Western blot analysis. Our data suggest a possible role for PRLR in the progression of cervical cancer.

Prolactin modulates cytokine production induced by culture filtrate proteins of M. bovis through different signaling mechanisms in THP1 cells.

The immunomodulatory functions of prolactin (PRL) are well recognized. Augmented PRL plasma levels were observed in patients with advanced tuberculosis (TB). Recently, we have reported that LPS and Mycobacterium bovis (M. bovis) induced differential expression of PRL receptor (PRLR) isoforms in THP-1 cells and bovine macrophages, respectively. The aim of this work was to determine whether PRL should be considered as a potential modulator of the signaling pathways and cytokine synthesis, induced by culture filtrate protein (CFP) from M. bovis in THP-1 monocytes. The THP-1 cells were stimulated with PRL (20ng/mL), M. bovis CFP (50µg/mL). PRLR as well as phosphorylated STAT3, STAT5, Akt1/2/3, ERK1/2 and p38 expression were evaluated by Western blot. IL1-ß, TNF-α, IL-6, IL-12, IL-8, and IL-10 concentrations were measured by ELISA. Our results demonstrated that the expression pattern of PRLR short isoforms is induced by M. bovis CFP. M bovis CFP induced phosphorylation of Akt2, ERK1/2, p38, STAT3, and STAT5 pathways. In turn, PRL only activated the JAK2/STAT3-5 signaling pathway. However, when combined both stimuli, PRL significantly increased STAT3-5 phosphorylation and downregulated Akt2, ERK1/2, and p38 phosphorylation. As expected, M. bovis CFP induced substantial amounts of IL1-ß, IL-6, TNF-α, IL-8, IL-12, and IL-10. However, the PRL costimulation considerably decreased IL1-ß, TNF-α, and IL-12 secretion, and increased IL-10 production. This results suggest that up-regulation of IL-10 by PRL might be modulating the pro-inflammatory response against mycobacterial antigens through the MAPK pathway.

Mycobacterium bovis infection in cattle induces differential expression of prolactin receptor isoforms in macrophages.

Prolactin receptor (PRLr) is a member of the cytokine receptor superfamily 1 showing tissue specific structural diversity. Expression of PRLr isoforms in lymphoid tissues has been associated with immunomodulatory function of prolactin. Bovine tuberculosis (bTB) is characterized by chronic inflammation caused by the persistent infection of lymphoid tissues with Mycobacterium bovis. To test the hypothesis of the influence of PRLr in the pathogenesis of bTB, the aim of this study was to identify PRLr isoforms expressed during bTB in different tissues and to analyze their association with the pathogenesis of bTB. We examined lymphoid and non-lymphoid tissues ex vivo from experimentally and naturally infected cattle, as well as from bTB-free cattle, by Western blot (WB) and immunohistochemistry (IH). In vitro, monocytes from exposed, infected, and healthy cattle were stimulated with M. bovis antigens and then analyzed by WB. To detect transcriptional levels of PRLr in macrophages (MØ) exposed to M. bovis, real time PCR was performed. WB revealed diversity of PRLr isoforms in tissues from infected cattle but not in tissues from bTB-free cattle. PRLr isoforms 100 kDa 75, 50 and 40 were found expressed in tissues of animals infected with M. bovis, while only the short isoform of 40 kDa correlated with the immunopathology and ability to infect MØ. We confirmed the synthesis of PRLr mRNA in MØ after M. bovis exposure and propose that molecular pathogen patterns of M. bovis might modulate inflammation during bTB through expression of the PRLr isoform in MØ.

High expression of prolactin receptor is associated with cell survival in cervical cancer cells.

BACKGROUND: The altered expression of prolactin (PRL) and its receptor (PRLR) has been implicated in breast and other types of cancer. There are few studies that have focused on the analysis of PRL/PRLR in cervical cancer where the development of neoplastic lesions is influenced by the variation of the hormonal status. The aim of this study was to evaluate the expression of PRL/PRLR and the effect of PRL treatment on cell proliferation and apoptosis in cervical cancer cell lines. RESULTS: High expression of multiple PRLR forms and PRLvariants of 60-80 kDa were observed in cervical cancer cell lines compared with non-tumorigenic keratinocytes evaluated by Western blot, immunofluorecence and real time PCR. Treatment with PRL (200 ng/ml) increased cell proliferation in HeLa cells determined by the MTT assay at day 3 and after 1 day a protective effect against etoposide induced apoptosis in HeLa, SiHa and C-33A cervical cancer cell lines analyzed by the TUNEL assay. CONCLUSIONS: Our data suggests that PRL/PRLR signaling could act as an important survival factor for cervical cancer. The use of an effective PRL antagonist may provide a better therapeutic intervention in cervical cancer.

The extrapituitary prolactin promoter polymorphism is associated with rheumatoid arthritis and anti-CCP antibodies in Mexican population.

Prolactin (PRL) is a hormone-cytokine that has been involved in autoimmunity due to its immunoregulatory and lymphoproliferative effects. It is produced by various extrapituitary sites including immune cells, under control of a superdistal promoter that contains a single nucleotide polymorphism -1149 G/T previously associated with rheumatoid arthritis (RA) susceptibility in European population. The aim of this study was to investigate the association of the extrapituitary PRL -1149 G/T promoter polymorphism with clinical parameters, clinical activity and disability indices in RA patients from Western Mexico and to analyze the PRL mRNA expression according to the PRL -1149 G/T promoter polymorphism in total leucocytes from RA patients and controls. We conducted a case-control study that included 258 RA patients and 333 control subjects (CS). The DNA samples were genotyped using the PCR-RFLP method and the PRL mRNA expression was determined by quantitative real time PCR. PRL serum levels and antibodies to cyclic citrullinated peptides (anti-CCP) were measured with ELISA. We found significant differences in the genotype (p=0.022) and allelic (p=0.046) distribution of the polymorphism between RA patients and control subjects. According to the dominant genetic model, there is an association between the T allele (GT+TT genotypes) and decreased RA susceptibility in comparison to the G allele carriers (GG genotype) (OR 0.64, 95% CI 0.45-0.92; p=0.011). The T allele carriers (GT+TT genotypes) had lower titers of anti-CCP antibodies in comparison to the G allele carriers (GG genotype) (median, 66 U/mL vs. 125 U/mL; p=0.03). Furthermore, the GG homozygotes had higher PRL mRNA expression in comparison to the GT heterozygotes, and this latter with respect to the TT homozygotes, in both groups (RA: 1>0.72>0.19; CS: 1>0.54>0.28). However, PRL serum levels were similar in both groups. Our results suggest that the PRL -1149 T allele is a genetic marker for decreased RA susceptibility and is associated with lower titers of anti-CCP antibodies in Mexican population. We also suggest influence of genotype upon PRL mRNA expression.

Increased expression of the prolactin receptor is associated with malignant laryngeal tumors.

The altered expression of the prolactin receptor (PRLR) has been associated with the development of various types of cancer, particularly breast, prostate and endometrial cancer. However, in laryngeal tumors, the expression of PRLR has not yet been documented. The aim of this study was to determine the expression and localization of PRLR in laryngeal cancer (LC) in comparison with recurrent respiratory papillomatosis (RRP). PRLR expression was analyzed in 48 paraffin-embedded tissues (18 RRP and 30 laryngeal cancer tissues) by immunoperoxidase staining. Furthermore, PRLR expression was evaluated in ten samples from each group by Western blot analysis and quantitative real-time PCR. PRLR was observed in all laryngeal tumors at different intensities. PRLR overexpression was significantly associated (P<0.005) with LC. The staining pattern was homogeneous, mainly cytoplasmic, and confined to the tumor area. We found increased expression of different isoforms in LC in comparison with RRP. Our results suggest a possible role of PRL/PRLR in the development of LC. PRLR may be useful as a target for further investigations in laryngeal tissues.

A microsatellite study of bovine solute carrier family 11 a1 (Slc11a1) gene diversity in Mexico in relation to bovine tuberculosis

Bovine tuberculosis, caused by Mycobacterium bovis, is a disease of socio-economic and public health importance and of significance to international trade regulation. Allelic variants of several genes have been implicated in the genetic susceptibility to tuberculosis in some human populations, but little is known in cattle. We surveyed 34 European, 18 Asian, 20 Creole and 23 hybrid bovines for polymorphisms of the bovine solute carrier family 11 a1(Slc11a1) gene, formerly known as natural resistance associated macrophage protein (Nramp1), gene by typing the cattle using two microsatellite loci closely linked to this gene. The microsatellites used were 311-22, located at the 3' untranslated region (3' UTR) of the Slc11a1 gene, and ARO28 situated about 0.6 cM upstream of the same gene Based on allele size in base pairs (bp) we determined five 311-223 locus variants (221, 223, 225, 227 and 229 bp) and 12 ARO28 loci. There was marked diversity and a very high level of heterozygosity in most of the cattle surveyed except the Europeans bovines and especially Holsteins in relation to the 3' UTR microsatellite locus.

Frequent alterations of the beta-catenin protein in cancer of the uterine cervix.

Cancer of the uterine cervix is still the leading cause of death among women with cancer in developing countries. Although infections with human papillomavirus are necessary, other molecular alterations that are needed at the cellular level for development of these tumors remain largely unknown. Beta-catenin is a key regulator located within the Wnt signaling cascade whose alterations constitute an important event in colon carcinogenesis. In many malignancies increased levels of the beta-catenin protein have been found, associated with its nuclear and/or cytoplasmic accumulation. To search for possible alterations of this pathway we examined the expression and localization of the beta-catenin protein in tumors from the uterine cervix and cell lines derived from them. Beta-catenin was found accumulated in the cytoplasm and/or nuclei of 12 out of 32 samples. In accordance, increased levels of this protein were observed in 9 out of 20 tumors analyzed. Importantly, PCR-SSCP and sequence analysis showed no mutations in exons 3, 4 and 6 of the beta-catenin gene. Our findings indicate that alterations of beta-catenin are frequent in these tumors and suggest that they may play an important role in the development of cancer of the uterine cervix. They also indicate that higher protein levels and abnormal localization may result from several different mechanisms.