Immunosuppression in hamsters with progressive visceral leishmaniasis is associated with an impairment of protein kinase C activity in their lymphocytes that can be partially reversed by okadaic acid or anti-transforming growth factor beta antibody

Progressive visceral infection of golden hamsters by Leishmania donovani amastigotes led to gradual impairment of the proliferative responses of their splenic or peripheral blood mononuclear cells (SPMC or PBMC, respectively) to in vitro stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io). Removal of macrophage-like adherent cells from SPMC or PBMC of infected animals (I-SPMC or I-PBMC) was earlier shown to restore almost completely their lymphoproliferative responses to PMA plus Io. The present study was directed to evaluate the status of protein kinase C (PKC), a molecule(s) known to play a key role in the lymphoproliferative process. Our results demonstrate that PKC activities (Ca(2+), phosphatidyl serine, and diacyl glycerol dependent) in the cytosolic fraction of untreated nonadherent I-SPMC or I-PBMC as well as in the membrane fraction of PMA-treated cells were decreased significantly relative to those for normal controls. However, removal of adherent cells from I-SPMC or I-PBMC and subsequent overnight in vitro cultivation of nonadherent cells (lymphocytes) resulted in significant restoration of PKC activity in the cytosolic or membrane fraction of untreated or PMA-treated cells, respectively. Partial, though significant, restoration of PKC activity could also be achieved in the membrane fraction of PMA-treated cells following overnight in vitro treatment of I-SPMC or I-PBMC with the Ser/Thr phosphatase inhibitor okadaic acid (OA) or an anti-transforming growth factor beta (anti-TGF-beta) neutralizing antibody. These results correlated well with the ability of OA or the anti-TGF-beta antibody to restore the lymphoproliferative response of I-SPMC or I-PBMC following stimulation with PMA plus Io. Interestingly enough, immunoblotting experiments failed to show any reduction in the level or translocation (following PMA treatment) of conventional PKC isoforms in the SPMC or PBMC of infected animals compared to those of normal controls. The results presented in this study suggest that the adherent cells generated in the SPMC or PBMC of infected animals exert a suppressive effect on the proliferative response of nonadherent cells (lymphocytes) which is likely to be mediated through the downregulation of the activation pathway involving PKC and its downstream molecules such as mitogen-activated protein kinases. Further, the observed suppression of PKC activity and subsequent lymphoproliferative responses can be attributed to alternations in the intracellular phosphorylation-dephosphorylation events. The relevance of these results is discussed in relation to the role of TGF-beta, levels of which are known to be elevated in visceral leishmaniasis.     

Evaluation of antileishmanial potential of Tinospora sinensis against experimental visceral leishmaniasis

The chemotherapeutic interventions against visceral leishmaniasis (VL) are limited and facing serious concerns of toxicity, high cost, and emerging drug resistance. There is a greater interest in new drug developments from traditionally used medicinal plants which offers unprecedented diversity in structures and bioactivity. With this rationale, ethanolic extract of Tinospora sinensis Linn and its four fractions were tested in vitro against promastigotes and intracellular amastigotes and in vivo in Leishmania donovani infected hamsters. Ethanolic extract exhibited an appreciable activity against promastigotes (IC₅₀ 37.6 ± 6.2 μg/ml) and intracellular amastigotes (IC₅₀ 29.8 ± 3.4 μg/ml). In hamsters, it resulted in 76.2 ± 9.2% inhibition at 500 mg/kg/day × 5 oral dose level. Among fractions, n-butanol imparted highest in vitro and in vivo activities. Ethanolic extract and butanol fraction also enhances reactive oxygen species (ROS) and nitric oxide (NO) release. The results indicate that T. sinensis may provide new lead molecules for the development of alternative drugs against VL.     

Dynamics of HIV-1 DNA level in highly antiretroviral-experienced patients receiving raltegravir-based therapy

To assess dynamics of HIV-1 DNA in highly antiretroviral (ARV)-experienced HIV-infected patients successfully treated with raltegravir (RAL)-containing therapy. Nineteen patients with virological failure whose ARV treatment was switched to a RAL-based salvage regimen with virological success were included (Group I). Ten patients in virological failure and responding to ARV salvage therapy not containing RAL were also included (Group II). The HIV-1 DNA level in peripheral blood mononuclear cells (PBMC) was assessed by real-time PCR at baseline, W12, W24, W36 or W48. In group I, a marked decrease in the HIV-1 DNA level was observed at W12 both in PBMC (median decrease = 0.38 log₁₀copies/10⁶PBMC; P < 0.001) and in CD4 T cells (0.85 log₁₀copies/10⁶CD4 T cells; P < 0.001). Plasma HIV-1 RNA decrease was correlated with HIV-1 DNA decrease expressed as copies/10⁶CD4 T cells (r = 0.55, P = 0.03). HIV-1 DNA level reached a steady state by W24. Thus, RAL-containing treatment in highly ARV-experienced patients was associated with a rapid HIV-1 DNA decrease, mainly in the circulating CD4 T cells compartment. Group II patients showed an early decrease in the HIV-1 DNA load until W12, which was 2.5-fold less pronounced in the CD4 T cells compartment than in the RAL-treated patients. The potent action of RAL-containing treatment observed in the CD4 T cells compartment may suggest a pronounced reduced inhibition in the pool of regenerating CD4 T cells on a RAL-based therapy.     

Transforming Growth Factor β and Immunosuppression in Experimental Visceral Leishmaniasis

Hamsters infected with Leishmania donovani develop a disease similar to human kala-azar. They present hypergammaglobulinemia, and their T cells do not respond to parasite antigens. This unresponsiveness has been primarily ascribed to defects in antigen-presenting cells (APCs), because these cells are unable to stimulate proliferation of parasite-specific T cells from immunized animals. In this study, we show that APCs (adherent spleen cells) from L. donovani-infected hamsters produce high levels of the inhibitory cytokine transforming growth factor β (TGF-β). Immunohistochemical studies with an anti-TGF-β monoclonal antibody (MAb) showed that this cytokine is abundantly produced in vivo by the spleen cells of infected animals. In addition, high levels of TGF-β are produced in vitro by infected hamster cells, either spontaneously or after stimulation with parasite antigen or lipopolysaccharide. Furthermore, in vivo-infected adherent cells obtained from spleens of L. donovani-infected hamsters caused profound inhibition of the in vitro antigen-induced proliferative response of lymph node cells from hamsters immunized with leishmanial antigens. Moreover, this inhibition was totally abrogated by the anti-TGF-β MAb. These results suggest that the immunosuppression observed in visceral leishmaniasis is, at least in part, due to the abundant production of TGF-β during the course of the infection.     

Peripheral blood mononuclear cell proliferative responses to soluble and particulate heat shock protein 65 in health and inflammatory bowel disease

Objectives: The aims of this study were to determine, in peripheral blood mononuclear cells (PBMC), whether particulate antigen triggers (i) an amplified cell proliferative response compared to soluble antigen and (ii) a dysfunctional response in cells derived from patients with chronic inflammation and specifically in those with inflammatory bowel disease (IBD). Subjects: Healthy volunteers (n = 17), inflammatory controls (n = 8) and patients with IBD (n = 17) were recruited from St Thomas’ and Guys’ Hospital, London, UK. Methods: Following optimisation of experimental conditions (0.1–10.0 μg/ml antigen), PBMC were stimulated with (i) 10.0 μg/ml recombinant soluble heat shock protein 65 (hsp 65) and (ii) 1.0 and 10.0 μg/ml hsp 65 conjugated to microparticles (0.5 μm diameter). PBMC proliferative responses were measured by ³H-Thymidine incorporation at day 5 and results compared between groups using unpaired t-test. Results: Conjugation to microparticles of low dose hsp 65 significantly increased overall proliferative responses by 2–11 fold compared to soluble antigen alone (p < 0.05). However, no specific PBMC proliferative dysregulation was noted in cells from subjects with IBD. Conclusions: Low dose antigen, in microparticulate form, leads to amplified cell proliferation in primary human cells, as showed previously in cell lines and animal studies. However there is no abnormal proliferative response in cells from subjects with IBD. Received 8 February 2006; returned for revision 7 March 2006; accepted by G. Wallace 25 October 2006     

Proliferating activity in columnar cell lesions of the breast

With the introduction of mammographic screening, columnar cell lesions (CCLs) are observed more and more frequently because they are often associated with microcalcifications. Until now, the proliferative activity of these lesions has not been previously evaluated. Ki67 index was performed by immunohistochemistry in CCLs without atypia [columnar cell change (CCC) n = 20 and columnar cell hyperplasia without atypia (CCH without atypia) n = 20], flat epithelial atypia (FEA DIN1A n = 20), low-grade intraductal carcinoma (DIN1C n = 20), high-grade intraductal carcinoma (DIN 2–3 n = 20). Adjacent terminal duct-lobular unit (TDLU) of normal breast tissue served as control. Ki-67 index is extremely low and close in CCLs without atypia (CCC mean 0.1% and CCH mean 0.76%) and paradoxically is lower than in normal TDLU (mean 2.4%) (p < 0.001). In the FEA, in comparison with normal TDLU and CCLs without atypia, the Ki67 is higher (mean 8.2%) (p < 0.001) but extremely close to those of DIN1C (mean 8.9%) (p = 0.6 NS). Lastly, the Ki67 index is higher in DIN 2–3 (mean 25.4%) than in CCLs without atypia and FEA (p < 0.001). CCLs are disparate lesions having in common cells with columnar configuration but different proliferative characteristics. These data represent findings of biological interest which could help us to better understand these controversial lesions.     

Immunomodulatory effects in Litomosoides sigmodontis-infected Mastomys coucha

The levels of parasite-specific IgG₁ and IgG₂ antibodies and mitogen-induced and parasite-specific proliferative T-cell responses were determined in Litomosoides sigmodontis-infected Mastomys coucha throughout an observation period of 400 days post infection (p.i.). These were compared with the respective reactions in animals that had been immunized with intrauterine stages/microfilariae of the parasite and in animals that had been challenged after immunization as determined at up to 60 days after challenge. IgG₁ antibodies to adult antigen developed early and reached a plateau at 120 days p.i., whereas IgG₂ antibodies were not found before day 60 p.i., increased with rising parasitemia, reached a plateau at 200 days p.i., and, in some animals, even became the predominant IgG subclass. Proliferative responses of spleen lymphocytes to concanavalin A (Con A) and lipopolysaccharides (LPS), but not Con-A-induced interleukin 2 (IL-2) production, were found to be suppressed in infected animals during patency as compared with uninfected controls. Spleen cells of infected animals showed a weak proliferative reaction to male antigen but were unresponsive to female and microfilarial antigen during prepatency and early patency. Subsequently, when microfilaremia decreased (200 days p.i.), continuously increasing responses to all antigens were observed. Immunized M. coucha developed specific IgG₁ and IgG₂ antibodies, and their spleen cells showed strong proliferative responses to the three L. sigmodontis antigens. Challenge infections down-regulated the proliferative responses of spleen cells to filarial antigens as early as during the prepatent phase of the challenge infection but supported existing IgG₁ and IgG₂ responses. Received: 1 September 1999 / Accepted: 24 September 1999     

Age-influenced population kinetics and immunological responses of <Emphasis Type="Italic">Leishmania donovani</Emphasis> in hamsters

Susceptibility of animals to infections depends upon various factors including sex and age of the host, which plays a pivotal role. In this communication, we have investigated the “intake” of Leishmania donovani infection in young (3–4 weeks old) and adult (15–16 weeks old) golden hamsters. The splenic parasite load in young hamsters on day 15 post infection (p.i.) was 54 ± 4 amastigotes/100 macrophage nuclei and increased to 106.3 ± 3.5 on day 30 p.i. However, adult group showed 2.2-(P < 0.001) and 1.75-fold (P < 0.001) lesser parasite burden on these days, respectively. But as the disease progresses further, differences in parasite burden become less significant, as revealed by comparable levels of parasite loads at 2 months p.i. Spleen weight measurements correspond to the above observations. In the young group, the levels of antileishmanial antibody rise two and 4.5 times on days 15 and 30 p.i., respectively, as compared to only 1.3 and 2.3 times increase in their respective adult counterparts. However, after 2 months of infection both groups recorded analogous (12-fold) rise in antibody levels. Both mitogenic and antigenic responses in adult hamsters were less suppressed compared to young hamsters on days 15 and 30 p.i. However, both groups exhibited highly suppressed cell-mediated immune (CMI) responses after 2 months of infection. These findings implicate that age of the host may influence the susceptibility and resistance to Leishmania infection.     

Comparison of T-cell responses in self-limiting versus progressive visceral Leishmania donovani infections in golden hamsters.

Leishmania donovani infection in golden hamsters was studied as a model for human kala-azar. After intradermal inoculation of L. donovani amastigotes, hamsters developed positive skin reactions (delayed-type hypersensitivity [DTH]) to parasite antigens and lymphoid cells from these hamsters proliferated to parasite antigens in vitro and transferred DTH reactivity to normal recipients. In contrast, hamsters infected by the intracardial route developed progressive visceral infections and failed to respond to skin test antigens. Spleen cells, lymph node cells, and peripheral blood lymphocytes (PBLs) from these hamsters were unresponsive to parasite antigens in vitro, and spleen cells failed to transfer DTH to normal recipients. Spleen cells, but not PBLs, displayed depressed responses to T-cell mitogens and also suppressed the proliferative response of cells from hamsters inoculated intradermally. Removal of adherent cells restored the capacity of spleen cells, but not PBLs, to respond to parasite antigens. The nonadherent population of these spleen cells also transferred DTH to normal recipients. The adherent suppressor cells, which have the characteristics of macrophages, appear to be localized to the spleen and are apparently not responsible for the failure of peripheral lymphoid cells to respond to antigen. These studies suggest that hamsters with visceral infections develop a population of antigen-reactive cells and that in the absence of suppression these cells may express functional activities, including the capacity to elicit DTH responses.     

Expression of the activation antigen CD69 predicts functionality of in vitro expanded peripheral blood mononuclear cells (PBMC) from healthy donors and HIV-infected patients

Gene therapy for AIDS necessitates harvest and expansion of PBMC from HIV-infected patients. We expanded PBMC from healthy blood donors and HIV-infected patients for up to 14 days using four expansion protocols: 3 days of phytohaemagglutinin (PHA) stimulation, continuous PHA stimulation, 3 days of stimulation with anti-CD3 and anti-CD28, and continuous stimulation with anti-CD3 and anti-CD28. Functionality of PBMC was evaluated prior to and after expansion using standard proliferation assay. Phenotype and lymphocyte subset activation defined by expression of CD69 and CD25 were determined using flow cytometry. PBMC from healthy donors and HIV-infected patients were readily expanded. The best expansion was obtained using stimulation for 3 days. After expansion, functionality of PBMC measured as proliferative response was partly conserved. PBMC expanded with stimulation for 3 days exhibited more preserved functionality than PBMC stimulated continuously (P < 0.03). The mean proliferative response in each of the four different expansion protocols correlated with the mean values of CD69 expression. The proliferative responses from patients and healthy donors expanded with PHA stimulation for 3 days correlated with CD69 expression on CD4 cells (r = 0.68, P < 0.01) and on CD8 cells (r = 0.59, P < 0.03). Furthermore, expression of CD69 reliably predicted which patients and donors had highly conserved functionality after in vitro expansion. Finally, PBMC expanded with PHA stimulation for 3 days were examined for apoptosis. Only a minor fraction was primed for apoptosis, and this fraction could be significantly reduced by addition of IL-2 to the culture medium (P < 0.05). In conclusion, the feasibility of expanding PBMC from HIV patients was demonstrated. Expanded PBMC had conserved functionality. Finally, after in vitro expansion, expression of the activation antigen CD69 reliably predicted functionality of PBMC.     

Mitochondrion-rich differentiated adenocarcinomas of the stomach: clinicopathological, immunohistochemical and electron microscopy study of nine cases

Pathologic and prognostic data of nine patients with mitochondrion-rich carcinomas (MRC) were compared retrospectively to data of 101 patients with conventional gastric adenocarcinomas. MRC was defined as a tumour composed predominantly, or entirely, of columnar adenocarcinoma cells with eosinophilic cytoplasm and a strong supranuclear immunoreactivity for antimitocondrial antibody. Electron microscopy confirmed supranuclear distribution of mitochondria in MRC while immunostaining pattern was irregular or absent in the remaining 101 cases. MRC exhibited a tubulopapillary or cribriform growth pattern with focal infiltration of neutrophils in the tumour stroma. Prominent necrosis was present including segmental and intraluminal “dirty necrosis”, while mitotic and ki-67 proliferative rates were low. MRC showed immunohistochemical findings compatible with gastric differentiation (CK7⁺/CK20⁻/CDX⁻).When MRC were compared with non-MRC carcinomas, tumour size (<4 cm vs >4 cm, P < 0.01), frequency of lymph node metastases (11% vs. 80%, P < 0.01), low stage (I, II) at diagnosis (100% vs. 56%, P < 0.01), Goseki’s group I (100% vs. 6%, P < 0.01), and better survival (0% vs. 70%, P < 0.01) differed significantly. Our results suggest that MRC of the stomach may be considered a low-grade malignancy with an excellent prognosis.     

Peripheral blood mononuclear cell responses to heat shock proteins and their derived synthetic peptides in juvenile idiopathic arthritis patients

Background Sequence homology and cross reactivity between microbial and human heat shock proteins (hsps) led to the concept that hsps might be involved in the etiopathogenesis of autoimmune diseases. Objective In our study we stimulated peripheral blood mononuclear cells (PBMC) of patients with juvenile idiopathic arthritis (JIA) and healthy controls with various hsp-derived peptides together with the whole molecules of corresponding hsp. Methods PBMC were cultured with recombinant human hsp60 (rh-hsp60), rh-hsp70, Mycobacterium bovis hsp65 (M.bovis hsp65), P562–571 human hsp60, P180–188 M. bovis hsp65, P450–463 human hsp70 and P545–554 cytokeratin derived synthetic peptides. Cell responses were measured after incorporation of ³H-thymidine and expressed as stimulation indices. Results and conclusion We found elevated proliferative response to rh-hsp60, M. bovis hsp65 and P562–571 human hsp60 derived peptide in patients with JIA polyarthritis. Significantly elevated proliferation to P180–188 M. bovis hsp65 was found in JIA lasting more than 2 years. None of the particular clinical characteristics (RF, ANA, HLA B27 and disease activity) seemed to be associated with hsp or hsp-derived synthetic peptide proliferative response in the JIA cohort. Received 26 September 2005; returned for revision 13 December 2005; accepted by G. Wallace 3 January 2006     

Protection of hamsters from amebic liver abscess formation by immunization with the 150- and 170-kDa surface antigens of Entamoeba histolytica

A monoclonal antibody, EH3015, prevents in vitro adherence of Entamoeba histolytica trophozoites to mammalian cells and inhibits amebic liver abscess formation in hamsters. By immunoaffinity chromatography with the monoclonal antibody, purified E. histolytica antigens with molecular masses of 150 and 170 kDa under non-reduced conditions were obtained. Hamsters were immunized with these antigens (group I) or with fractions further purified by polyacrylamide gel electrophoresis (group II). Pooled immune sera from the two groups inhibited in vitro amebic adherence to Chinese hamster ovary cells by 98% at 1:10 dilutions. The immunized hamsters were challenged by the intrahepatic injection of E. histolytica trophozoites. Complete protection from abscess formation was observed in 38% of hamsters in group I and 67% in group II, whereas all control hamsters inoculated only with adjuvant developed amebic liver abscesses. In the immunized hamsters, the abscesses in the two groups were significantly smaller than in the controls. These results demonstrate that the E. histolytica antigens are possible vaccine candidates for amebiasis. Received: 10 August 2000 / Accepted: 5 September 2000     

The proliferation-associated nuclear protein Ki-67 in the bovine system: partial characterisation and its application for determination of the proliferation of Theileria-infected bovine cells

Theileria annulata-infected bovine cells as well as mitogen-stimulated bovine peripheral blood mononuclear cells (PBMC) express a proliferation-associated nuclear protein equivalent to the human Ki-67 protein. In analogy to the human system, the expression of the bovine Ki-67 protein is restricted to proliferating cells only, since (a) Ki-67 expression paralleled [³H]-thymidine incorporation in concanavalin A (Con A)-stimulated bovine PBMC, (b) Ki-67 was not detectable in quiescent bovine cells, and (c) Ki-67 expression in Theileria-infected cells is related to the presence of the parasites within the cytoplasm of the host cells; upon treatment with the theilericidal drug buparvaquone the parasites are destroyed and the cells cease to proliferate and to express the Ki-67 protein. Western-blot analysis of lysates of proliferating bovine cells revealed that the prototype monoclonal antibody Ki-67 and the new equivalent antibody MIB-1 detected one prominent protein band with an apparent molecular weight of 430 kDa. Two cDNA clones (pUC18.B1.Ki-67 and pUC18.B2.Ki-67) were isolated from a λgt11 cDNA library of T. annulata-infected bovine cells by immunoscreening with the monoclonal antibody MIB-1. Comparison of these cDNA sequences with those of the human Ki-67 protein revealed 60–70% identity. Within the “Ki-67 motif”, identity proved to be 80% at the amino acid level. The remarkable identity between bovine and human Ki-67 proteins suggests that MIB-1 can be used as a marker for cell proliferation in animal research. In this context we could identify proliferating cells in lymph nodes of Theileria-infected animals and, furthermore, we could distinguish between infected and uninfected proliferating cells using MIB-1 and an antiserum against a recombinant parasite protein designated SA²⁸⁸. Received: 6 October 1998 / Accepted: 22 January 1999     

Changes in peripheral blood lymphocyte subpopulations and parasite-specific antibody responses in Trypanosoma evansi infection of sheep

This paper reports on changes in the lymphocyte composition of the peripheral blood in sheep infected with Trypanosoma evansi. In addition, parasite-specific IgG₁ and IgM antibody responses were monitored using a double-sandwich enzyme-linked immunosorbent assay (ELISA) technique. Eight sheep were infected with 2 × 10⁶ T. evansi TREU 2143. The infection was characterised by chronicity and ended in self-cure in two of the sheep. These two sheep were designated group A, whereas the other six sheep, which remained parasitaemic until treated, were designated group B. Analysis of the peripheral blood lymphocytes (PBLs) by indirect immunofluorescence staining and flow cytometry revealed significant alterations in the numbers of T- and B-cell subsets detected in all infected sheep. In group A, whereas the numbers of CD8⁺ cells decreased, CD4⁺ cells showed marginal decreases, remaining at or above pre-infection figures and resulting in increase in the CD4:CD8 ratio. In group B, CD8⁺ cells showed few marginal decreases, being at or above pre-infection figures most of the time, whereas CD4⁺ cells decreased significantly from day 26 post infection (p.i.) such that the CD4:CD8 ratio decreased. Infection also resulted in significant increases (P < 0.001) as of day 26 p.i. in circulating B-cells in group B as shown by the numbers of sIg⁺, CD45R⁺, CD1⁺ and major histocompatibility complex (MHC) II⁺ cells. The increases, however, were moderate and biphasic in group A. T. evansi-specific IgM and IgG₁ antibody isotypes were detected in all infected sheep, but their levels were significantly higher in group A than in group B (IgM P <0.05; IgG₁ P <0.01). In addition, although an initially higher level of IgM response was subsequently replaced by a higher level of IgG₁ response in group A, this was never the case in group B until after drug treatment. Received: 16 May 1998 / Accepted: 17 June 1998     

Immune responses of the domestic fowl to Dermanyssus gallinae under laboratory conditions

There appear to be few reports in the literature regarding the host–poultry red mite (Dermanyssus gallinae) immunological relationship, despite the negative impact D. gallinae can have on both bird welfare and egg production, as well as its potential to act as a reservoir of zoonotic infections. The current study investigated the effect of either continuous infestation (CI) for 22 days or repeated infestation (RI) for four 24-h periods 7 days apart with D. gallinae on humoral immunity (IgM and IgY) and Th1/Th2 cytokine mRNA expression in peripheral blood mononuclear cells (PBMC) compared to non-infested controls. Serum IgY levels and IgM concentration were significantly higher in CI than RI and control birds although Th1 and Th2 mRNA expression in PBMC did not differ significantly between groups. D. gallinae appeared to modify reproductive behaviour and progeny survival following successive feeding events. In the RI group, the proportion of eggs/mite was significantly higher (P < 0.05) after first infestation than later infestations while larval/nymphal mortality was significantly higher (P < 0.05) after the first two infestations than later infestations. These data suggest that D. gallinae might adopt a feeding strategy of minimal host interference while D. gallinae could determine host immune status via nymphal/larval survival rates. Further research is required to better understand the host immunomodulation or avoidance strategy of D. gallinae as well as whether the mite is able to determine host immunocompetence perhaps using progeny survival.     

Tyrosine phosphatase SHP-1 is expressed higher in multisystem than in single-system Langerhans cell histiocytosis by immunohistochemistry

Langerhans cell histiocytosis (LCH) is a proliferative disorder of Langerhans cell (LC)-like CD1a-positive cell (LCH cell) with unknown causes. LCH consists of two subtypes: single-system LCH (LCH-SS) with favorable prognosis and multisystem LCH (LCH-MS) with poor prognosis. LCH has been indicated as a neoplastic disorder from monoclonal characteristics of LCH cells. This study aimed to investigate an expression of tyrosine phosphatase SHP-1 in LCH, since its expression levels were variously reported in many tumors, overexpression in ovarian cancers (a candidate oncoprotein), and downregulation by methylation in gastric cancers, prostate cancers, malignant lymphomas, and leukemias (a putative tumor suppressor). By immunohistochemistry (IHC), the SHP-1 expression in LCs and LCH cells was compared in LCH (two subtypes: LCH-SS = 21, LCH-MS = 12), dermatopathic lymphadenopathy (DLA) (n = 9) and normal epidermal LCs (n = 3) near LCH lesion. IHC results were analyzed semiquantitatively using a Photoshop software. The mean intensity score (IS) of DLA, LCH-SS, LCH-MS, and LCs were 47, 100, 139, and 167 (in arbitrary unit), respectively. The IS had significant differences among LCH-SS, LCH-MS, and DLA (p < 0.01). SHP-1 is expressed significantly higher in LCH-MS than in LCH-SS. SHP-1 can be a progression marker of LCH. SHP-1 is also useful for differential diagnosis between LCH in lymph nodes and DLA.     

Clinical evaluation of canine mast cell tumor treatment between combined vinblastine and prednisolone and single prednisolone

Thirty three dogs having cutaneous mast cell tumors (MCT) were divided into two groups; group 1 had 23 dogs that were treated with vinblastine and prednisolone and group 2 had 10 dogs that were treated with prednisolone. Evaluation of the 33 pre- and post-treated tissue samples was performed on their clinical stages, histopathological features, expression of proliferative markers such as argyrophilic nucleolar organizer regions (AgNORs), proliferative cell nuclear antigen (PCNA) and Ki-67 and clinical response. All cases were diagnosed as MCT grade II without post-treatment changes in histopathological grade. An increase of uniform nucleus, stroma but a decrease of cytoplasm, the number of visible nucleolus, intralesional vascularization, eosinophilic aggregation, and the mean of mitotic index (pretreatment = 1.6 cells/HPF, post-treatment = 1.1 cells/HPF) was observed in the post-treated samples of both groups. With regard to the clinical evaluation, 18 dogs (78.2%) were partially responsive and the rest (21.8%) were stable in group 1 while five dogs (50%) were partially responsive, three dogs (30%) were stable, and the remaining two dogs (20%) were progressive in group 2. The median survival time of the group 1 cases was 101 days and for the group 2 cases was 175 days. In addition, mean ± standard deviations of AgNORs (dots/cell), PCNA (%), and Ki-67 (%) were 1.83 ± 0.4, 18.67 ± 9.25, and 6.86 ± 7.23 in the pretreatment group 1 and 1.59 ± 0.3, 12.4 ± 7.15, and 1.9 ± 1.35 in the post-treatment group 1, respectively. Mean ± standard deviations of AgNORs, PCNA and Ki-67 were 1.83 ± 0.42, 18 ± 20.01 and 6.74 ± 5.42 in the pretreatment group 2 and 1.67 ± 0.28, 6.28 ± 5.59 and 2.3 ± 1.55 in the post-treatment group 2, respectively. All proliferative markers decreased differently statistically after treatment (P < 0.05). In conclusion, the MCT dogs treated with vinblastine and oral prednisolone and single oral prednisolone were shown to have a decrease in histopathological malignancy characteristics which included AgNORs, PCNA, Ki-67 indices.     

Immune response pattern of the popliteal lymph nodes of dogs with visceral leishmaniasis

The present study aimed to estimate the cell response and parasite load in the popliteal lymph nodes of dogs with visceral leishmaniasis (VL), comparing these findings with the clinical staging of the disease. From the necropsy, 33 dogs were classified as symptomatic (S), asymptomatic (A), or oligosymptomatic (O). Cytology and histopathology were used to determine any presence of microscopic lesions and immunohistochemistry, for parasite load. Dog hyperimmune serum was used as the primary antibody. The inflammatory infiltrate in lymph nodes consisted of macrophages and plasmocytes. The granulomas invaded the trabecular and sinusoid regions and sometimes compressed the lymphocytes of the cortical region (atrophy) and medullary cord cells. Parasite load intensity was unrelated to the density of the macrophages infiltrating the lymph node. Significant differences in parasite load (P < 0.05) were observed between the three groups of infected dogs. Follicular hyperplasia of the cortical region occurred among A and O, while follicular atrophy predominated among S. The parasite load was the greatest among S, followed by O. It can be concluded that, regardless of clinical condition, the most evident cell response consisted of macrophages and plasmocytes. Lymphoid atrophy was observed among animals with intense granulomatous reaction and high parasite load, such as among the symptomatic dogs (P < 0.05). Likewise, the oligosymptomatic dogs also presented high density of parasites in the lymph nodes. Thus, we can confirm that dogs with clinical manifestations of VL have an immune system that is less effective for controlling infection by Leishmania chagasi, thereby favoring parasite multiplication.     

Prognostic significance of the Fas-receptor/Fas-ligand system in cervical squamous cell carcinoma

We studied whether Fas-receptor (Fas-R; CD95) expression, single-nucleotide polymorphisms (SNPs) in the Fas promoter region, and/or Fas-ligand (Fas-L) production could determine individual susceptibility to cervical cancer progression. The clinicopathologic features of 38 patients with cervical squamous carcinomas (22 stage I, 8 stage II, and 8 stage III+) were reviewed and related with: (a) Fas-R expression by immunohistochemistry; (b) Fas-R SNPs at -670 and -1377 locations by restriction fragment length polymorphism and DNA sequencing; and (c) Fas-L expression by immunohistochemistry. Overall and disease-free survival curves showed significant differences in relation to stage (p < 0.001). Fas-R was identified in 20 of 38 (52.6%) tumors without statistical differences in survival, stage, or Fas-L overproduction. Fas-R GG genotype was more common than expected in advanced tumors (p = 0.065). The Fas-R-1377A allele and AA genotype were unrelated with survival, stage, or Fas-R expression. Fas-L overproduction was detected in 20 of 38 (52.6%) tumors; it was more frequent in advanced-stage tumors and was inversely related to survival (p = 0.03) and decrease in host inflammatory response (p = 0.01). Fas-R expression by tumor cells seems unrelated to stage or lymphoid infiltrate. Tumor production of Fas-L may represent an attempt to destroy the host’s lymphocytic reaction.