Physicochemical properties of intact fungal cell wall determine vesicles release and nanoparticles internalization.

Our previous microscopic observations on the wet mount of cultured Candida yeast showed release of large extracellular vesicles (EVs) that contained intracellular bacteria (∼500-5000 nm). We used Candida tropicalis, to examine the internalization of nanoparticles (NPs) with different properties to find out whether the size and flexibility of both EVs and cell wall pores play role in transport of large particles across the cell wall. Candida tropicalis was cultured in N-acetylglucoseamine-yeast extract broth (NYB) and examined for release of EVs every 12 h by the light microscope. The yeast was also cultured in NYB supplemented with of 0.1%, 0.01% of Fluorescein isothiocyanate (FITC)-labelled NPs; gold (0.508 mM/L and 0.051 mM/L) (45, 70 and 100 nm), albumin (0.0015 mM/L and 0.015 mM/L) (100 nm) and Fluospheres (0.2 and 0.02%) (1000 and 2000 nm). Internalization of NPs was recorded with fluorescence microscope after 30 s to 120 min. Release of EVs mostly occurred at 36 h and concentration of 0.1% was the best for internalization of NPs that occurred at 30 s after treatment. Positively charged 45 nm NPs internalized into >90% of yeasts but 100 nm gold NPs destroyed them. However, 70 nm gold and 100 nm negatively-charged albumin were internalized into <10% of yeasts without destroying them. Inert Fluospheres either remained intact on the surface of yeasts or became degraded and internalized into ∼100% of yeasts. Release of large EVs from the yeast but internalization of 45 nm NPs indicated that flexibility of EVs and cell wall pores as well as physicochemical properties of NPs determine transport across the cell wall.

Detection of peptidoglycan in yeast as a marker for the presence or abundance of intracellular Helicobacter pylori and Staphylococcus.

Peptidoglycan (PG) was targeted as the marker for bacterial occurrence inside yeast. Detection of only few bacteria in old and new generations of yeast raised the question of how yeast controls the abundance of its intracellular bacteria. One gastric C. tropicalis that showed concurrence of H. pylori and Staphylococcus 16S rDNA was stained for assessing the viability of intracellular bacteria. Fluorescein isothiocyanate (FITC)-labeled anti-PG monoclonal antibody (APGMAb) was used for detection of PG inside yeast by direct immunofluorescence. APGMAb-coated magnetic beads were used for separation of bacteria from disrupted yeasts. Bead-bound bacteria were separated, fixed, stained, and examined by scanning electron microscope (SEM). Bead-bound bacteria were cultured and identified by amplification and sequencing of 16S rDNA. Fluorescence microscopy demonstrated occurrence of few live bacteria inside yeast cells. FITC- APGMAb interacted with PG of intracellular bacteria, appearing as few green spots in mother and daughter yeast cells. Interestingly, PG fragments were also detected in the exterior of yeast cells. SEM observations showed separated bead-bound bacilli and cocci. Culture of Staphylococcus was positive. Sequencing results confirmed identity of separated bacteria as H. pylori and Staphylococcus. PG detected inside yeast may have belonged to H. pylori, Staphylococcus or any other intracellular bacteria that coexisted in yeast as its microbiome. Detection of only few intracellular bacteria in old and new generations of yeast as well as PG fragments in their exterior suggested that yeast controls the abundance of its intracellular bacteria at low rate by hydrolysis and exporting of PG.

Weissella confusa with thermostable ß-hemolytic exopolysaccharide.

Consuming cooked meat contaminated with bacteria that carry thermostable hemolytic exopolysaccharide (ESP), could lead to severe diseases. Culture of a 5- h boiled sample of meat goulash on blood agar showed growth of a gram positive rod-shaped, mucoid and hemolytic bacterium. Biochemical tests and amplification of 1500 bp product of 16S rDNA and sequencing revealed bacterial identity as Weissella confusa. After 1 h boiling of bacterial suspension, they were alive and hemolytic, increased in volume and aggregated. After 8 h boiling of bacterial suspension with coverslip, live bacteria showed hemolysis, clustered and adhered to coverslip. Bacterial bacteriocin and hemolytic activities remained unchanged upon autoclaving. Purified bacterial EPS retained hemolytic activity after autoclaving. Boiling contaminated meat had no negative impact on viability of heat-stable W. confusa and its hemolytic EPS. Thermostable hemolytic EPS protected W. confusa from excessive heat. Hygienic practice in butcheries and kitchens are necessary to eliminate bacterial contaminants.

Coniochaeta fungus benefits from its intracellular bacteria to form biofilm and defend against other fungi.

During cultivation of a gastric fungus, Coniochaeta polymorpha, growth of Nocardia colonies on top of the fungal culture raised the question whether bacteria originated from inside of fungus. In this study, the likelihood of intracellular origin of bacteria as well as interaction of two microorganisms was assessed. Fluorescence and electron microscopy showed occurrence of several bacterial cells in fungal cytoplasm. A thick biofilm was observed on the surface of co-culture compared with thin one on bacterial and none on fungal monocultures. Field emission scanning electron microscopy (FESEM) micrographs of co-culture showed a dense network of fungal and bacterial cells embedded in a slime-like layer. Dual cultures revealed antagonistic activity of both fungus and bacteria against three Candida species. These findings indicate that Nocardia isolate identified in this study originated from the inside of fungus C. polymorpha. Intracellular bacteria could benefit the fungal host by producing a rigid biofilm and an antifungal compound.

Sugar-Rich Foods Carry Osmotolerant Yeasts with Intracellular Helicobacter Pylori and Staphylococcus spp.

BACKGROUND Sugar-rich foods are of the main components of daily human meals. These foods with high sugar and low water content kill bacteria. However, osmotolerant yeasts survive and multiply. The aim of this study was to examine the occurrence of intracellular Helicobacter pylori (H. pylori) and Staphylococcus spp. in yeast isolates from sugar-rich foods. METHODS Thirty-two yeast isolates from fresh fruits, dried fruits, commercial foods, and miscellaneous foods were identified by the sequencing of amplified products of 26S rDNA. Fluorescence microscopy and LIVE/DEAD bacterial viability kit were used to examine the occurrence of live bacteria inside the yeast's vacuole. Immunofluorescence assay was used to confirm the identity of intracellular bacteria as H. pylori and Staphylococcus . Polymerase chain reaction (PCR) was used for the detection of 16S rDNA of H. pylori and Staphylococcus in the total DNA of yeasts. RESULTS Yeasts were identified as members of seven genera; Candida, Saccharomyces, Zygosaccharomyces, Pichia, Meyerozyma, Metschnikowia, and Wickerhamomyces. Intravacuolar bacteria were stained green with a bacterial viability kit, revealing that they were alive. Immunofluorescence assay confirmed the identity of intracellular H. pylori and Staphylococcus spp. PCR results revealed that among the 32 isolated yeasts, 53% were H. pylori -positive, 6% were Staphylococcus -positive, 18.7% were positive for both, and 21.8% were negative for both. CONCLUSION Detection of H. pylori - and Staphylococcus -16S rDNA in yeast isolates from dried fruits, and commercial foods showed the occurrence of more than one kind of endosymbiotic bacterium in yeasts' vacuoles. While the establishment of H. pylori and Staphylococcus in yeast is a sophisticated survival strategy, yeast serves as a potent bacterial reservoir.

Helicobacter pylori release from yeast as a vesicle-encased or free bacterium.

BACKGROUND: Yeast has been suggested as a potent reservoir of H. pylori that facilitates bacterial spread within human populations. What mechanism ensures effective H. pylori release from yeast? Here, H. pylori release from yeast as a vesicle-encased or free bacterium was studied. MATERIALS AND METHODS: Liquid culture of Candida yeast was examined by light, fluorescence and transmission electron microscopy methods to observe the released vesicles. Vesicles were isolated and examined by TEM. Immunogold labeling was used for detection of H. pylori-specific proteins in vesicles' membrane. Free bacterial cells, released from yeast, were separated by immunomagnetic separation and observed by field emission scanning electron microscopy (FESEM). DNA of bead-bound bacteria was used for amplification of H. pylori-16S rDNA. Viability of bead-bound bacteria was examined by live/dead stain and cultivation on Brucella blood agar. RESULTS: Microscopic observations showed that vesicles contained bacterium-like structures. Thin sections showed release of vesicle-encased or free bacterium from yeast. Immunogold labeling revealed occurrence of H. pylori proteins in vesicles' membrane. FESEM showed attachment of H. pylori cells to magnetic beads. Sequencing of 521 bp PCR product confirmed the identity of bead-bound H. pylori. Live/dead staining showed viability of bead-bound H. pylori but the result of culture was negative. CONCLUSIONS: Escape of intracellular H. pylori from yeast as a membrane-bound or free bacterium indicates that H. pylori uses safe exit mechanisms that do not damage the host which is the principle of symbiotic associations. In human stomach, certain conditions may stimulate yeast cells to release H. pylori as a vesicle-encased or free bacterium.

Yeast engineered translucent cell wall to provide its endosymbiont cyanobacteria with light.

In this study, relationship between translucent property of yeast cell wall and occurrence of cyanobacteria inside the yeast vacuole was examined. Microscopic observations on fruit yeast Candida tropicalis showed occurrence of bacterium-like bodies inside the yeast vacuole. Appearance of vacuoles as distinct cavities indicated the perfect harvesting of light by the yeast's cell wall. Transmission electron microscopy observation showed electron-dense outer and electron-lucent inner layers in yeast cell wall. Cyanobacteria-specific 16S rRNA gene was amplified from total DNA of yeast. Cultivation of yeast in distilled water led to excision of intracellular bacteria which grew on cyanobacteria-specific medium. Examination of wet mount and Gram-stained preparations of excised bacteria showed typical bead-like trichomes. Amplification of cyanobacteria-specific genes, 16S rRNA, cnfR and dxcf, confirmed bacterial identity as Leptolyngbya boryana. These results showed that translucent cell wall of yeast has been engineered through evolution for receiving light for vital activities of cyanobacteria.

Proton Pump Inhibitor-Treated H. pylori Adjust Cell Envelope Fatty Acid and Cholesterol Content to Survive.

BACKGROUND: Proton pump inhibitors (PPIs) with lipophilic nature may interact with lipid components of H. pylori cell membrane, disrupting cell structure and viability. In this study, the effect of PPIs on fatty acid and cholesterol components of H. pylori cell membrane was assessed. METHODS: One H. pylori isolate was treated with 1X and 2X MICs (µg/mL) of lansoprazole (LPZ: 8 and 16) and pantoprazole (PAN: 128 and 256) in brain heart infusion broth plus serum. Treated H. pylori was cultured on brucella blood agar (BBA) and tetrazolium egg yolk agar (TEYA). Bacterial cells stained with Live/Dead kit were examined by fluorescent microscopy. Fatty acid and cholesterol contents of treated H. pylori were measured by gas chromatography. RESULTS: PPI-treated H. pylori did not grow on BBA but grew on TEYA. Fluorescent microscopy showed H. pylori stained red. Analyses showed high frequency of saturated fatty acids, C14:0, C16:0 and C18:0. Among unsaturated fatty acids, C18:1 and C18:2c were increased, while five were eliminated and five were synthesized de novo. Cholesteryl-6-O-tetradecanoyl-α-D- glucopyranoside was detected as the only glycosylated cholesterol in treated H. pylori. Growth of PPI-treated H. pylori on cholesterol-rich TEYA showed that occurrence of cholesterol can reverse the growth inhibition by PPIs. Red- bacilli form of H. pylori showed dye entry through damaged cell membrane without lysis. CONCLUSION: Incorporation of lipophilic PPI into H. pylori cell membrane disrupted lipids and inhibited growth. However, H. pylori adjusted the defected membrane by replacing the lipid components and resisted lysis.

Excision of endosymbiotic bacteria from yeast under aging and starvation stresses.

Although infrequent in our laboratory, growth of bacterial colonies has been observed on top of the purified cultures of yeasts. In this study, the likelihood of bacterial excision from yeast under aging and starvation stresses was assessed using 10 gastric and 10 food-borne yeasts. Yeasts were identified as members of Candida or Saccharomyces genus by amplification and sequencing of D1/D2 region of 26S rDNA. For aging stress, yeasts were cultured on brain heart infusion agar supplemented with sheep blood and incubated at 30 °C for 3-4 weeks. For starvation stress, yeasts were inoculated into distilled water and incubated similarly. After seven days, starved yeasts were cultured on yeast extract glucose agar, incubated similarly and examined daily for appearance of bacterial colonies on top of the yeast's growth. Outgrowth of excised bacteria was observed on top of the cultures of 4 yeasts (Y1, Y3, Y13 and Y18) after 3-7 days. The excised bacteria (B1, B3, B13 and B18) were isolated and identified at the genus level according to their biochemical characteristics as well as amplification and sequencing of 16S rDNA. B1 (Arthrobacter) were excised from Y1 (Candida albicans) upon aging and B3 (Staphylococcus), B13 (Cellulomonas) and B18 (Staphylococcus) were excised from their respective yeasts; Y3 (Candida tropicalis), Y13 (Saccharomyces cerevisiae) and Y18 (Candida glabrata) upon starvation. DNA from yeasts was used for detection of 16S rDNA of their intracellular bacteria and sequencing. Amplified products from yeasts showed sequence similarity to those of excised bacteria. Under normal conditions, yeast exerts tight control on multiplication of its intracellular bacteria. However, upon aging and starvation the control is no longer effective and bacterial outgrowth occurs. Unlimited multiplication of excised bacteria might provide yeast with plenty of food in close vicinity. This could be an evolutionary dialogue between yeast and bacteria that ensures the survival of both partners.

Sequestration inside the yeast vacuole may enhance Helicobacter pylori survival against stressful condition.

Vacuole of eukaryotic cells, beyond intracellular digestion plays additional roles such as storage of nutrients that provide favorable conditions for bacterial survival. In this study, occurrence of H. pylori inside the vacuole of Candida yeast was studied and the role of vacuolating cytotoxin A (VacA) in constructing the vacuole was discussed. One gastric Candida yeast was used for Live/Dead stain and fluorescence in situ hybridization (FISH) with universal bacterial probe. Yeast total DNA was used for amplification of full-length bacterial 16S rDNA as well as H. pylori-specific 16S rDNA and vacA alleles. Vacuoles were isolated from yeast cells and stained with fluorescent yeast vacuole membrane marker MDY-64. DNA extracted from vacuoles was used for amplification of H. pylori-specific 16S rDNA. Fluorescent microscopy showed occurrence of viable bacteria inside the vacuole of intact Candida yeast cells. FISH showed intracellular bacteria as fluorescent spots inside the vacuole of mother and daughter yeast cells, suggesting bacterial transmission to next generations of yeast. Sequencing of amplified products of bacterial 16S rDNA and amplification of H. pylori 16S rDNA and vacA confirmed the identity of intracellular bacteria as H. pylori. Isolated vacuoles were stained with membrane-specific marker and H. pylori 16S rDNA was amplified from their DNA content. Results of this study suggest yeast vacuole as a specialized niche for H. pylori. It appears that sequestration inside the vacuole may enhance bacterial survival.

Increased Urine Interleukin-17 and Interleukin-22 Levels in Patients With Candidal Urinary Tract Infection.

INTRODUCTION: Candiduria is common in the hospitalized patients. This study aimed to quantify interleukin (IL)-17 and IL-22 levels in urine of candiduric patients. MATERIALS AND METHODS: A case-control study was conducted on inpatients at Hashemi Nejad Kidney Center. Thirty-four patients were identified with Candida species in their urine samples (> 103 colony-forming units per milliliter and presence of Candida species only). Urine samples with concomitant infections were excluded. Thirty-four patients with negative direct examination and culture were included as the control patients. Interleulin-17 and IL-22 levels were measured in the lyophilized and nonlyophilized urine. The relevant cytokine titers of the two groups were compared, and the association of cytokine elevation and candiduria was investigated. RESULTS: The majority of the candiduric patients were from the intensive care and urology units of women. Only 4 patients (11.7%) manifested fever and dysuria. Massive leukocyturia was observed in 4 patients. Candida glabrata was the most commonly isolated species (44%). Levels of the urine IL-17 and IL-22 were significantly elevated in the candiduric patients, when compared to the noncandiduric controls. While an increased IL-17 level was significantly associated with candiduria (odds ratio, 1.09; 95% confidence interval, 1.003 to 1.17; P = .04), an increased IL-22 level was not. The results showed that lyophilized urine samples maximized the detection power of urinary cytokines. CONCLUSIONS: Our results indicated that direct examination, fungal urine culture, and investigation of urine IL-17 and IL-22 levels are useful tools for diagnosis of Candida urinary tract infection.

Increased levels of CD4(+) and CD8(+) T cells expressing CCR1 in patients developing allograft dysfunction; a cohort study.

BACKGROUND: Leukocyte infiltration into the graft has pivotal effects on kidney transplantation outcome. The present study sought to determine whether the expression of sequential chemokine receptors on CD4(+) and CD8(+) T cells in human renal allograft can predict clinical episodes. METHODS: Blood samples from 52 consecutive renal transplant patients were evaluated at the time of transplantation and at three times (2, 90 and 180days) after transplantation to analyze the expression of CCR1 and CXCR3 on CD4(+) and CD8(+) T cells by flowcytometry. A total of 30 biopsies, including protocol biopsy (n=24) and cause biopsy (n=6), were investigated according to the Banff criteria. RESULTS: The mean percentage of CD4(+) and CD8(+) T cells expressing CCR1 was significantly increased in patients with allograft dysfunction (n=25) (p=0.006, p=0.004). The mean fluorescence intensity of CXCR3 on CD4(+) and CD8(+) T cells were found to be significantly higher in graft dysfunction than that in well-functioning grafts. (p<0.001, p=0.007). Receiver Operating Characteristic (ROC) Curve Analysis showed that the calculated AUC was 0.86 at the third month for CD4(+)CCR1(+) and CD8(+)CCR1(+) (p<0.001). Multiple logistic regression analysis showed that an increase in CD4(+) expressing CXCR3 leads to a lower risk of graft dysfunction (OR=0.37), while an increase in CD8(+) expressing CCR1 results in a higher risk of graft dysfunction (OR=3.66). CONCLUSION: During renal transplantation, CD4(+) and CD8(+) T cells expressing CCR1 were increased in patients who developed graft dysfunction. These findings may prospectively predict allograft dysfunction, and help elucidate the underlying pathogenic mechanisms.

Hippocampal DHCR24 down regulation in a rat model of streptozotocin-induced cognitive decline.

The DHCR24 (24-dehydrocholesterol reductase) gene codes a multifunctional protein which consists of enzymatic, antioxidant, and anti-apoptotic activities. It exists in almost all neurons and protects the neural cells against amyloid ß toxicity. Several studies have shown the down regulation of DHCR24 in Alzheimer's disease. We examined the time profile of DHCR24-mRNA alteration in an animal model of streptozotocin (STZ)-induced cognitive impairment. The DHCR24 mRNA levels of hippocampus and cognitive impairment were evaluated at 7, 14, and 21 days after intracerebroventricular (ICV)-STZ/Saline administration. DHCR24 expression was down regulated at 14 and 21 days after ICV-STZ administration. The decrease in expression of DHCR24 preceded the onset of the cognitive impairment. These results suggest the potential relation between DHCR24 expression and cognitive impairment.

Dynamic changes of regulatory T cell and dendritic cell subsets in stable kidney transplant patients: a prospective analysis.

INTRODUCTION: We aimed to identify immune status of the stable kidney allografts from the point of some cellular changes that may occur after transplantation. MATERIALS AND METHODS: This study considered 57 patients with no rejection during the 6 months after transplantation. Flow cytometric frequencies of circulatory CD4+CD25+FoxP3+ and CD8+CD28- regulatory T cells (Treg) as well as myeloid dendritic cells type 1 (MDC1) and type 2 (MDC2) and plasmacytoid dendritic cells (PDC) were measured before transplantation and 2 weeks and 1, 3, and 6 months after transplantation. Using adjusted model of repeated measure analysis, we assessed the influence of different parameters on different cell subsets. RESULTS: The mean number of Tregs and PDCs decreased 2 weeks after transplantation and then increased as they reached their values before transplantation within a few months after transplantation. The mean MDC1s increased during 2 weeks and then decreased to its before-transplantation values within 6 months. The frequency of Tregs (r = 0.90) and MDC1s (r = 0.75) at month 3 could strongly predict their frequencies at month 6. Different variables including family relationship between donor and recipient, glomerular filtration rate, and human leukocyte antigen antibody mismatch did not change the frequency of different cell subsets during the time. CONCLUSIONS: The dynamism and circulatory changes in the frequency of Tregs and PDCs are opposite to MDCs after kidney transplantation. We describe these changes in a group of patients with stable graft; however, our study does not render any idea in patients with unstable or rejecting grafts.

Elevated CSF and plasma microparticles in a rat model of streptozotocin-induced cognitive impairment.

Alzheimer's disease (AD), can be described as a vascular disorder, is characterized by endothelial and platelet activation. One feature of activated cells is loss of lipid asymmetry, and membrane blebbing which cause microparticle (MP) formation. MPs increased under many pathological states and little information is available relating to their changes in AD. The purpose of this work was to characterize the time course of the endothelial-derived microparticles (EMPs) and platelet-derived microparticles (PMPs) alteration after intracerebroventricular (ICV) injection of streptozotocin (STZ). Rats were injected bilaterally with ICV-STZ/Saline, cerebrospinal fluid (CSF) and plasma EMPs (Annexin V(+) CD61(-)CD144(+)) and PMPs (Annexin V(+) CD61(+)CD144(-)) were analyzed with flow cytometry at 2 h, 4 h, 24 h, 4 days, 7 days, 14 days and 21 days after ICV-STZ/Saline administration. Cognitive impairment, malondialdehyde (MDA) level of hippocampus, plasma serotonin, and serum S100B were also assessed. We showed the elevation of CSF and plasma level of EMPs and PMPs, which may represent a proinflammatory and prothrombotic status. These alterations were simultaneous with the hippocampal MDA rise, plasma serotonin increment, and S100B decrement, 7 days after ICV-STZ administration and precede the onset of cognitive impairment. Understanding the profile of MP changes in CSF or plasma as biomarkers from tissues undergoing activation or damage, may be helpful in prediction or early diagnosis of AD.

Conversion of calcineurin inhibitors with mammalian target of rapamycin inhibitors after kidney transplant.

One way to overcome chronic allograft nephropathy induced by calcineurin inhibitors in immunosuppression protocols for organ transplants is to replace such inhibitors with mammalian target of rapamycin inhibitors, which are not clinically nephrotoxic because they have better renal function. If patients tolerate replacement, there could be a clear preference for mammalian target of rapamycin inhibitors as a maintenance immunosuppressant after renal transplant. This replacement could be sufficient if it were used for a certain time after calcineurin inhibitors. This review considers the conversion effects of calcineurin inhibitors with mammalian target of rapamycin inhibitors from the view point of kidney function during different periods after a kidney transplant.

CCR7+ central and CCR7- effector memory CD4+ T cells in human cutaneous leishmaniasis.

PURPOSE: The profile of central (=T(CM)) and effector (=T(EM)) memory CD4(+) T cell subsets and the possible role as surrogate markers of protection is studied in the volunteers with history of cutaneous leishmaniasis (HCL). METHODS: Profile of T cell subsets based on CCR7/CD45RA expressions and phenotypic changes after soluble Leishmania antigen (SLA) stimulation were analyzed. Then, sorted CD4(+)CD45RO(-)CD45RA(+) naïve T, CD4(+)CD45RO(+)CD45RA(-)CCR7(-) T(EM,) CD4(+)CD45RO(+)CD45RA(-)CCR7(+) T(CM) subsets were cultured with SLA for proliferation, cytokine production and intracellular cytokine assays. RESULTS: In the HCL and control volunteers, the mean frequencies of CD4(+)CD45RA(+)CCR7(+) naïve T cells and CD4(+)CD45RA(-)CCR7(-) T(EM) cells were higher than the other subsets before culture. Frequency of naïve T cells and CD4(+)CD45RA(-)CCR7(+) T(CM) cells was significantly decreased (P=0.01 for naïve T and P<0.05 for T(CM) cells) and frequency of T(EM) cells was significantly increased after SLA stimulation compared to before culture (P<0.001). By CFSE labeling, CD4(+)CD45RO(+)CD45RA(-)CCR7(+) T(CM) cells showed more proliferation potential than CD4(+)CD45RO(+)CD45RA(-)CCR7(-) T(EM) cells. Stimulation of the T(EM) cells in HCL volunteers induced a significantly higher IFN-γ production (P=0.04) with higher number of intracellular IFN-γ positive cells (P=0.032) than the same cells from controls. A significantly higher number of T(CM) cells produced IL-2 in HCL volunteers compared with controls (P<0.05). Most of the intracellular IFN-γ positive T(EM) cells were proliferating CFSE-dim populations (P<0.05). CONCLUSIONS: A combination of Leishmania-reactive IFN-γ producing CD4(+)CD45RO(+)CD45RA(-)CCR7(-) T(EM) and Leishmania-reactive IL-2 producing CD4(+)CD45RO(+)CD45RA(-)CCR7(+) T(CM) are identified in individuals with history of CL which might play a role in protective recall immune response against Leishmania infection.

Downregulation of IL-17-producing T cells is associated with regulatory T cell expansion and disease progression in chronic lymphocytic leukemia.

Little is known about the immunobiology of interleukin-17 (IL-17)-producing T cells and regulatory T cells (Treg) in chronic lymphocytic leukemia (CLL). In this study, the frequencies of Th17, Tc17, and CD39(+) Treg cells were enumerated in peripheral T cells isolated from 40 CLL patients and 15 normal subjects by flow cytometry. Our results showed a lower frequency of Th17 and Tc17 cells in progressive (0.99 ± 0.12 % of total CD3(+)CD4(+) cells; 0.44 ± 0.09 % of total CD8(+) cells) compared to indolent patients (1.57 ± 0.24 %, p = 0.042; 0.82 ± 0.2 %, p = 0.09) and normal subjects (1.78 ± 0.2 %, p = 0.003; 0.71 ± 0.09 %, p = 0.04). Decrease in IL-17-producing T cells was associated with CD39(+) Treg cells expansion. Variation of IL-17-producing cells and Treg cells in indolent and progressive patients was neither associated to the expression levels of Th1- and Th2-specific transcription factors T-bet and GATA-3 nor to the frequencies of IFN-γ and IL-4-producing CD4(+) T cells in a selected number of samples. Additionally, suppressive potential of CD4(+) Treg was similar in CLL patients and normal subjects. Our data indicate that progression of CLL is associated with downregulation of IL-17-producing T cells and expansion of Treg cells, implying contribution of these subsets of T cells in the progression of CLL.

Phenotyping of circulating CD8⁺ T cell subsets in human cutaneous leishmaniasis.

Recovery from CL is usually accompanied with long-lasting protection and induction of strong immune response. The phenotypes, generation and maintenance of central (=T(CM)) and effector (=T(EM)) memory T cell subsets in human leishmaniasis are not well known. Profile of T cell subsets were analyzed on peripheral CD8⁺ T cells from volunteers with history of cutaneous leishmaniasis (HCL). In HCL and control groups, mean frequencies of CCR7⁺CD45RA⁺CD8⁺ naïve and CCR7⁻CD45RA⁻CD8⁺ T(EM) cells were higher than other subsets before culture, but after stimulation with soluble Leishmania antigen, the frequency of naïve T cells was significantly decreased and the frequency of T(EM) cells was significantly increased. T(EM) phenotype composed the highest portion of proliferating Carboxy Fluorescein diacetate Succinimidyl Ester (CFSE)-dim population which was significantly higher in HCL volunteers than in control group. Stimulation of isolated CD8⁺ memory T cells, but not naïve T cells, from HCL volunteers induced a significantly higher IFN-γ production compared with that of healthy controls. Intracellular IFN-γ staining provided the same result. Memory population is shown to be responsible for Leishmania-induced IFN-γ production. Leishmania-reactive proliferating T(EM) cells were identified as the most frequent subset which may play a role in recall immune response and protection against Leishmania infection.

Combination effect of PectaSol and Doxorubicin on viability, cell cycle arrest and apoptosis in DU-145 and LNCaP prostate cancer cell lines.

The effect of PectaSol on Dox (Doxorubicin) cytotoxicity in terms of apoptosis and cell cycle changes in PCa (prostate cancer) cell lines (DU-145 and LNCaP) has been investigated. Combination of PectaSol and Dox resulted in a viability of 29.4 and 32.6% (P<0.001) in DU-145 and LNCaP cells. The IC50 values decreased 1.5-fold and 1.3-fold in the DU-145 and LNCaP cells respectively. In the DU-145 cells, combination of PectaSol and Dox resulted in a reduction in p27 gene and protein expression (P<0.001). In LNCaP cells, this combination increased p53, p27 and Bcl-2 expression. Treatment with both drugs in DU-145 cells led to an increase in sub-G1 arrest (54.6% compared with 12.2% in Dox). In LNCaP cells, combination of the drugs led to an increased in G2/M arrest (61.7% compared with 53.6% in Dox). Based on these findings, progressive cytotoxicity effect of Dox and PectaSol together rapidly induce cell death in DU-145 through apoptosis and in LNCaP cells through cell cycle arrest (G2/M arrest).