The Salivary Mycobiome Contains 2 Ecologically Distinct Mycotypes.

A broad range of fungi has been detected in molecular surveys of the oral mycobiome. However, knowledge is still lacking on interindividual variability of these communities and the ecologic and clinical significance of oral fungal commensals. In this cross-sectional study, we use internal transcribed spacer 1 amplicon sequencing to evaluate the salivary mycobiome in 59 subjects, 36 of whom were scheduled to receive cancer chemotherapy. Analysis of the broad population structure of fungal communities in the whole cohort identified 2 well-demarcated genus-level community types (mycotypes), with Candida and Malassezia as the main taxa driving cluster partitioning. The Candida mycotype had lower diversity than the Malassezia mycotype and was positively correlated with cancer and steroid use in these subjects, smoking, caries, utilizing a removable prosthesis, and plaque index. Mycotypes were also associated with metabolically distinct bacteria indicative of divergent oral environments, with aciduric species enriched in the Candida mycotype and inflammophilic bacteria increased in the Malassezia mycotype. Similar to their fungal counterparts, coexisting bacterial communities associated with the Candida mycotype showed lower diversity than those associated with the Malassezia mycotype, suggesting that common environmental pressures affected bacteria and fungi. Mycotypes were also seen in an independent cohort of 24 subjects, in which cultivation revealed Malassezia as viable oral mycobiome members, although the low-abundance Malassezia sympodialis was the only Malassezia species recovered. There was a high degree of concordance between the molecular detection and cultivability of Candida, while cultivation showed low sensitivity for detection of the Malassezia mycotype. Overall, our work provides insights into the oral mycobiome landscape, revealing 2 community classes with apparently distinct ecologic constraints and specific associations with coexisting bacteria and clinical parameters. The utility of mycotypes as biomarkers for oral diseases warrants further study.

Hydrothermal Ethanol Flames in Co-Flow Jets.

Results on the autoignition and stabilization of ethanol hydrothermal fames in a Supercritical Water Oxidation (SCWO) reactor operating at constant pressure are reported. The flames are observed as luminous reaction zones occurring in supercritical water; i.e., water at conditions above its critical point (approximately 22 MPa and 374 °C). A co-flow injector is used to inject fuel (inner flow), comprising an aqueous solution ranging from 20 %-v to 50 %-v ethanol, and air (annular flow) into a reactor filled with supercritical water at approximately 24.3 MPa and 425 °C. Results show hydrothermal fames are autoignited and form diffusion flames which exhibit laminar and/or turbulent features depending upon flow conditions. Two orthogonal camera views are used; one providing a backlit shadowgraphic image of the co-flow jet and the other providing color images of the flame. In addition, spectroscopic measurements of flame emissions in the UV and visible spectrum are discussed.

Serum catalytic Iron: A novel biomarker for coronary artery disease in patients on maintenance hemodialysis.

Cardiovascular disease is the leading cause of morbidity and mortality in maintenance hemodialysis (MHD) patients. We evaluated the role of serum catalytic iron (SCI) as a biomarker for coronary artery disease (CAD) in patients on MHD. SCI was measured in 59 stable MHD patients. All patients underwent coronary angiography. Significant CAD was defined as a > 70% narrowing in at least one epicardial coronary artery. Levels of SCI were compared with a group of healthy controls. Significant CAD was detected in 22 (37.3%) patients, with one vessel disease in 14 (63.63%) and multi-vessel disease in eight (36.36%) patients. The MHD patients had elevated levels of SCI (4.70 ± 1.79 µmol/L) compared with normal health survey participants (0.11 ± 0.01 µmol/L) (P < 0.0001). MHD patients who had no CAD had SCI levels of 1.36 ± 0.34 µmol/L compared with those having significant CAD (8.92 ± 4.12 µmol/L) (P < 0.0001). Patients on MHD and diabetes had stronger correlation between SCI and prevalence of CAD compared with non-diabetics. Patients having one vessel disease had SCI of 8.85 ± 4.67 µmol/L versus multi-vessel disease with SCI of 9.05 ± 8.34 µmol/L, P = 0.48. In multivariate analysis, SCI and diabetes mellitus were independently associated with significant CAD. We confirm the high prevalence of significant CAD in MHD patients. Elevated SCI levels are associated with presence of significant coronary disease in such patients. The association of SCI is higher in diabetic versus the non-diabetic subgroup. This is an important potentially modifiable biomarker of CAD in MHD patients.

Is carbon dioxide a safe and good alternative for diatrizoate meglumine as a contrast in digital subtraction angiography?

Contrast-induced nephropathy is well-known sequelae of iodinated contrast (diatrizoate meglumine). Carbon dioxide (CO(2)) can be used as an alternative contrast agent. The aim of this study was to compare the renal injury and the quality of images of aortogram using iodinated contrast versus CO(2) using digital subtraction angiography (DSA). This prospective randomized study was done in 29 healthy dogs using DSA aortogram. Dogs were randomly assigned to receive iodinated contrast or CO(2). 6-F pigtail catheter was introduced via femoral artery approach to perform aortogram under general anesthesia. Serum creatinine (S.Cr.) and urinary enzymes, namely: N-acetyl D-glucosaminidase (NAG), alanine aminopeptidase (AAP), and gamma glutamyl transferase (GGT), were measured before and 48 hours after aortogram. There was no change in S.Cr. in both the groups. Significantly more enzymuria was seen following iodinated contrast than CO(2). Enzymuria pre and postaortogram following the iodinated contrast was GGT: 14.9 +/- 5.92 vs. 26.2 +/- 15.1 (P = 0.001), NAG: 1.63 +/- 0.90 vs. 3.6 +/- 2.14 (P = 0.0001), and AAP: 1.51 +/- 0.75 vs. 3.38 2.41 (P = 0.001), and in the CO(2) group was GGT: 15.5 +/- 4.9 vs. 21.1 +/- 9.04 (P = 0.02), NAG: 2.12 +/- 1.06 vs. 3.82 3.27 (P = 0.08), and AAP: 1.28 +/- 0.76 vs. 2.51 +/- 1.72 (P = 0.03). More than 50% increase over the preprocedural value was significantly less following CO(2). Images obtained with iodinated contrast were superior to those with CO(2,) however, the quality of image with CO(2) was adequate for delineation of the renal artery and major branches. Both iodinated contrast and CO(2) cause significant enzymuria. More severe enzymuria (>50% increase) was seen significantly less with the use of CO(2). Quality of images is better with iodinated contrast.

Gene expression profiling of lymphomas.

The functional phenotype of a cell results from the simultaneous action of many thousands of genes, which until recently could not be assessed using standard molecular biological techniques. Indeed, molecular genetics and cellular biology inadequately explain the molecular physiology of normal and diseased cells and provide a fragmented view of the role of various genes and their products. Recent advances in techniques of large-scale gene expression allow simultaneous study of thousands of genes of interest in a specific tissue/tumor of interest, and the ability to identify expression signatures associated with functional phenotypes. The application of gene expression profiling to lymphomas has already led to identification of distinct expression signatures associated with a germinal center cell and activated B-cell phenotype, and to the differentiation of tumor cells based on these stages of development. The differentiation of two stages of developmental arrest in large B-cell lymphomas suggests that this subtype is comprised of two diseases, albeit of similar histologic and immunophenotypic character, which were shown to have dramatically different outcomes following chemotherapy. Important information will also be obtained through the association of genes of unknown activity with functional cellular phenotypes and expression signatures, possibly leading to identification of new genes involved in lymphomagenesis and new targets for treatment.

Immunoregulatory pathways in pregnancy.

Murine pregnancy is characterized by transient thymic atrophy and splenomegally. Several laboratories are investigating the immunoregulatory mechanisms during pregnancy, and the majority of these studies are primarily focused on the immunological changes either in the uterus or the thymus and not much information is available on the immunological changes in the spleen that result in transient splenomegally. An attempt has been made in this review to understand the significance of thymic atrophy, splenomegally and local immune changes in the uterus to understand the overall immunomodulatory mechanisms in pregnant mother. The most significant change which occurs soon after mating is the infiltration of immune cells such as macrophages and gammadelta-T cells into the uterus indicating that the mother's immune system detects the presence of foreign antigens in the reproductive tract. The sensitized cells appear to migrate to the secondary lymphoid organs including the spleen. The microenvironment in the spleen is conducive for the cell-cell contact and generation of immune response. The major changes that occur in the spleen are, the induction of T-cell dependent B-cell response on day-1 post-coitum (P.C.), generation of antibody producing B-cells on day-3 and also proliferation of CD8+ T-cells that peaks on day-3 of pregnancy. The weight of the spleen reaches a peak on day-10 in mice. Thereafter, on day-15 of pregnancy, lymphocyte apoptosis is seen in the spleen indicating the deletion of peripheral sensitized cells. This results in decrease in spleen weight to that of normal non-pregnant mice. The decrease in thymic weight after day-5 pregnancy was associated with the increased apoptosis of cortical thymocytes. This perhaps is due to negative selection of self-reactive thymocytes. Our studies have demonstrated that the pregnancy associated monoclonal antibodies react with antigens of sperm indicating that the mother's immune system recognizes and responds to the constituents of the semen to produce non-precipitating asymmetric auto antibodies (NPAA) or blocking antibodies which have favourable effects on pregnancy. It is postulated that the mother's immune response could be directed to some antigens of sperm along with some conserved antigens such as heat shock proteins (HSP) that are present both in sperm and in the mother. It may be speculated that after the initial priming to some conserved antigens of sperm and due to the presence of similar antigens in the mother, these activated clones are eliminated both in the primary and secondary lymphoid organs to prevent autoimmunity in the mother during pregnancy.

Multiple myeloma and immune thrombocytopenia.

Immune thrombocytopenia (ITP) is frequently encountered in patients with lymphoproliferative disorders. However this is only rarely reported in patients with multiple myeloma. We describe three cases who presented initially with the clinical manifestations of ITP but were subsequently found to have multiple myeloma. Platelet count increments to standard treatment modalities for ITP were observed in all three patients with transient or partial response. The importance of recognizing the immune mediated thrombocytopenia in patients with myeloma and the implications of this combination are discussed.

The role of LFA-1, Mac-1, ICAM-1 and Ia in the induction of Th-2 type of immune response in spleen during murine syngeneic pregnancy.

Murine syngeneic pregnancy is characterized by the transient splenomegaly at mid gestation. Recent studies from our laboratory have indicated the initiation of T-cell dependent B-cell response in the spleen during early pregnancy (Hegde and Nainan 1998). Present studies were carried out to understand the role of cell adhesion and MHC class II (Ia) molecules in the induction of Th-2 type of response in the spleen of pregnant mouse. Immunochemical localization of ICAM-1, LFA-1, Mac-1 and Ia in spleen have been carried out at different stages of pregnancy and formation of cell clusters and natural cell adhesion assay with splenocytes were carried out on day 1 (D1) pregnancy and compared with control. Upregulation of ICAM-1, LFA-1, Mac-1 and Ia was observed during early pregnancy. This coincided with the formation of germinal centers (GC) and Th2 type of interleukins in spleen as reported earlier. Increased expression of cell adhesion and Ia molecules during early pregnancy provides additional evidence for the systemic shift to Th2 type of immune response in syngeneic murine pregnancy.

T helper-2 type of interleukins with de novo germinal center formation in the spleen during murine pregnancy.

PROBLEM: Murine pregnancy is characterized by transient thymic atrophy and splenomegaly. Although some reports on the thymus are available, little is known about the role of the spleen. In the present study, sequential changes in the spleen have been evaluated during murine syngeneic pregnancy. METHOD OF STUDY: Formation of germinal centers (GCs), immunoglobulin positive (Ig+) cells and interleukin (IL)-2, IL-4, and IL-6 were immunolocalized in the spleen during pregnancy and compared with a control group of virgin mice. RESULTS: Initiation of T-cell dependent B-cell response with the induction of peanut agglutinin (PNA+) GCs correlated with decrease in IL-2 and maintenance of IL-4 and IL-6 on day-1 pregnancy followed by an increase in Ig+ cells. CONCLUSION: The immune response observed in the syngeneically pregnant mother is not directed to paternal major histocompatibility complex antigens. Present studies demonstrate for the first time the role of the spleen in initiating a T-cell dependent B-cell response with a shift of systemic immune response from T helper 1 to T helper 2 type.

Bcl-2 and c-myc expression, cell cycle kinetics and apoptosis during the progression of chronic myelogenous leukemia from diagnosis to blastic phase.

Chronic myelogenous leukemia (CML) has a progressive course but little is known about the biologic characteristics of disease progression. This study was designed to assess the changes in cell proliferative characteristics, apoptosis, the expression of the bcl-2 and c-myc genes between the time of initial diagnosis and entrance into the blastic phase of the disease. We observed that the rate of cell proliferation decreased and the cell death rate did not significantly change as the disease accelerated. The level of bcl-2 expression was significantly higher in accelerated/blastic phase cells than in the chronic phase cells in the population as a whole, however, the bcl-2 expression level did not change in blast cell subpopulation. c-myc Expression was significantly higher in the blast cell subpopulation of accelerated/blastic phase than in that of earlier phases of the disease. In conclusion, the characteristics of CML cells, namely proliferation rate, c-myc and bcl-2 change during the course of the disease. It is possible that the change in c-myc expression plays a causative role in evolution of the blastic phase from the chronic phase.

Immunochemical characterization and antifertility effect of an androgen independent antigen of spermatozoa identified by a monoclonal antibody (D7G3).

Monoclonal antibody, (mAb) D7G3, directed to human spermatozoa and cross-reactive with mouse and rat spermatozoa was found to be a sperm agglutinating antibody. It reacted with antigen present on the post acrosomal region of human spermatozoa and acrosomal region of mouse spermatozoa. The antigen reacting with mAb D7G3 was localized in mouse testicular germ cells and Sertoli cells. It was also localized in the epithelium and spermatozoa of the caput, corpus and cauda epididymides. In addition, the antigen was detected in the epithelial cells and secretions of the prostate and seminal vesicle. The mAb D7G3 reacted with a band of molecular mass 16 kDa in testicular and epididymal sperm and protein preparations of ventral prostate. In the seminal vesicle, an additional band of molecular mass 36 kDa was also identified. The effect of castration on the expression of proteins was studied in the rat. Immunohistochemical localization and Western blotting experiments showed that the antigens identified by the mAb D7G3 was detectable in the epididymis, ventral prostate and seminal vesicle after two weeks of castration. In the seminal vesicle, three additional bands were identified by mAb D7G3 following castration. The expression of this antigen throughout spermatogenesis and sperm maturation indicated that it may have an important biological function. A polyclonal antiserum directed to the 16 kDa mouse epididymal sperm protein was raised in rabbits. Passive immunization of female mice with this antibody caused a significant reduction in fertility only if administered 24 hours prior to mating, indicating that the antibody inhibited a pre-fertilization event by inhibiting sperm function.

Hyperferritinaemia in the absence of iron overload.

BACKGROUND: Serum ferritin is normally a marker of iron overload. Ferritin genes are sited at chromosomes 19 and 11. Regulation of ferritin synthesis involves an interaction between an iron regulatory protein (IRP) and part of the ferritin mRNA designated the iron regulatory element (IRE). A disorder of ferritin synthesis resulting in hyperferritinaemia in the absence of iron overload has been described recently. PATIENTS AND METHODS: Hyperferritinaemia in the absence of iron overload was detected in a patient who was investigated for possible haemochromatosis. Serum iron, transferrin saturation, and ferritin concentration were studied in 11 members of this patient's family from three generations. Eight members had DNA samples analysed by direct cycle sequencing of the 5' untranslated region of the L ferritin gene. RESULTS: Six of the family members studied had serum ferritin concentrations greater than 900 micrograms/l. However, serum iron and transferrin saturation were normal in these subjects who all had evidence of cataracts. Three affected family members who had genetic studies of the L ferritin gene on chromosome 19 had an A to G point mutation which was not found in unaffected members. CONCLUSIONS: There was complete concordance between a mutated IRE, cataracts, and hyperferritinaemia in three generations of this family. This family study confirms the finding that hereditary hyperferritinaemia in the absence of iron overload is an autosomal dominant inherited disorder.

The effects of 13-cis retinoic acid and interferon-alpha in chronic myelogenous leukemia cells in vivo in patients.

The effects of the administration of a 3-day course of 13-cis retinoic acid in combination with interferon a [RA/IFN] on the leukemia cells was measured in vivo in 43 patients with chronic myelogenous leukemia. The administration of RA/IFN was associated with a significant fall in the white blood cell count of patients with chronic-phase disease and with a fall in the percentage S-phase cells in CML patients regardless of the stage of their leukemia. In two thirds of the patients studied the administration of RA/IFN was also associated with an increase in marrow apoptosis. The cytokine combination also suppressed bcl-2 and myc expression in a minority of patients and such expression appears to be associated with response to a treatment regimen which includes RA/IFN. These studies are the first to directly assess the effects of the combination of RA/IFN on chronic myelogenous leukemia cells in vivo in patients. These effects, if seen in other malignant diseases, could account for the therapeutic benefit which has been associated with the administration of this combination of biological agents to patients with malignant disease.

Accuracy of immunohistochemistry and flow cytometry in estimating the proportion of leukemic cells in S phase in chronic myeloid leukemia patients.

We compared the proportion of S phase cells in bone marrow and peripheral blood samples obtained from 17 patients with chronic myeloid leukemia (CML). Before sampling all patients received a one hour i.v. infusion of iododeoxyuridine (IdUrd). The proportion of S phase cells was studied by immunohistochemistry (IHC) in bone marrow biopsies, and by flow cytometry (FCM) in bone marrow aspirates and peripheral blood samples. The IdUrd labelling index (LI) in bone marrow biopsy sections (27.5 +/- 1.8%) was significantly higher than the proportion of IdUrd labelled cells in bone marrow aspirate (15.1 +/- 2.0%). The percentage of S phase cells in peripheral blood was approximately the same as that in the aspirate (12.4 +/- 1.3%) and was correlated with that of bone marrow aspirate indicating a high degree of the aspirate dilution by peripheral blood. It is likely that the differences in % S phase cells in the aspirate and the biopsy result from this dilution. Estimates of the % S phase cells in the peripheral blood study by IHC and FCM were essentially the same. Samples labelled for one hour in vitro resulted in 1.5 fold higher LI than the same samples labelled in vivo. We conclude that estimates of the 8% S phase cells in the bone marrow of patients with CML should be made by infusing patients with IdUrd or BrdUrd with immunohistochemical evaluation of a marrow biopsy. Additionally in vitro labelling is not reflective of the percent S phase cells in vivo in patients.

Epididymal sperm maturation proteins.

Mammalian spermatozoa are not motile when they leave the testis. They have to undergo a complex maturation process to be able to fertilize in vivo. The maturation changes of mammalian sperm membrane have been extensively studied using lectins and antibodies. Some of these antigens have been purified and cloned. The interaction of secreted proteins with sperm membranes and acquisition of sperm motility as essential steps for spermatozoa to be fertile are well documented. The role of these epididymal maturation proteins in infertility and the possibility of using these antigens for immunocontraception are discussed in this review.

Antigenic stimulation and blocking of pregnancy in mice by an anti-idiotypic antibody to progesterone.

PROBLEM: The present study was carried out to see if the anti-idiotypic (anti-Id) antibody (Ab2) to progesterone could mimic the immunogenicity of the steroid hormone by giving rise to antiprogesterone antibodies (Ab3) and whether these antiprogesterone antibodies (Ab3) were biologically active. METHOD: Twenty virgin female Balb/C mice were actively immunized with the anti-Id antibody (Ab2). The antiprogesterone antibody (Ab3) titres in the serum were determined and the animals were used for fertility studies. In the passive immunization studies Balb/C female mice were injected i.p. with 100 micrograms of anti-Id antibody to see its effect on pregnancy. RESULTS: The actively immunized animals when mated showed 80% reduction in their fertility rate. The duration of infertility (20-121 days) in these animals could be directly correlated with the concentration of antiprogesterone antibody (Ab3). The anti-Id antibody (Ab2) blocked pregnancy in 80% of the passively immunized mice. CONCLUSION: The studies show that anti-Id antibody to progesterone could mimic the immunogenicity of progesterone and give rise to antiprogesterone antibodies (Ab3). The anti-Id antibody successfully blocked pregnancy in mice both after active and passive immunization.

Prognostic significance of myeloperoxidase containing blast cells in acute myelogenous leukaemia.

Fifty three newly diagnosed patients of de novo acute myelogenous leukaemia (AML) received treatment consisting of remission induction with daunorubicin 60 mg/m2 on day one and continuous infusion of cytosine arabinoside 200 mg/m2/day over 24 h from day one to 7. Thereafter patients in complete remission received consolidation chemotherapy with two identical courses. Complete remission (CR) could be achieved in 40 patients (75.5%). Seven patients (13.2%) died with complications during aplasia phase following remission induction therapy while six patients (11.3%) had resistant disease. Twenty seven patients (67.5%) developed relapse while eight patients (15.1%) continue to remain in complete remission ranging from 51 to 68 months (median 62.5). The projected event free survival and disease free survival at 60 months is 15 per cent (SE + 11.9%) and 21 per cent (+6%) respectively. Evaluation of the prognostic significance of pretherapy characteristics showed that infection at presentation and low number of myeloperoxidase (MPO) containing blasts affected the achievement of complete remission adversely on univariate analysis. Similarly age at diagnosis, of more than 45 yr, total leucocyte count of 50,000/cumm or more and low number of MPO containing blasts affected the remission duration (disease free survival) adversely on univariate analysis. On multivariate analysis, MPO positivity of blast cells, remained the only significant independent characteristic. High MPO positivity affected the remission duration favourably (P < 0.01). Patients with high MPO positivity also achieved CR with one induction cycle in 32 out of 40 instances while only 2 out of 5 patients with low MPO positivity, achieved CR with one chemotherapy cycle (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Human macrophage colony-stimulating factor levels are elevated in pregnancy and in immune thrombocytopenia.

Plasma macrophage colony-stimulating factor (M-CSF) levels were measured by enzyme immunosorbent assay (ELISA) using horse and rabbit polyvalent antibodies raised against human M-CSF purified from urine (hM-CSF). Plasma M-CSF levels in nonpregnant female controls were 364 +/- 69 U/mL (mean +/- SD, n = 20). Pregnancy results in significant elevation of circulating M-CSF levels (541 +/- 164 U/mL, n = 46, P < .0005). M-CSF levels were increased by 28 weeks' gestation and did not increase further in later pregnancy. M-CSF levels were also measured in 20 female controls before and after commencing on the oral contraceptive pill. There was no effect of the contraceptive pill on plasma M-CSF levels (364 +/- 69 U/mL before v 373 +/- 66 U/mL after commencing on the pill). In 28 nonpregnant patients with untreated immune thrombocytopenic purpura, (ITP), plasma M-CSF levels were significantly increased (797 +/- 402 U/mL, n = 28, v 364 +/- 69 U/mL in controls, N = 20, P < .0005). Pregnant ITP patients had higher levels of plasma M-CSF (929 +/- 327 U/mL, n = 25) than nonpregnant patients, but this difference was not significant. Elevated levels of M-CSF in ITP may reflect activation of the reticuloendothelial system (RES), which could result in positive feedback to increase the destruction of platelets. The increase in M-CSF associated with pregnancy could contribute to the exacerbation of latent ITP in pregnancy.

Localization and detection of ovarian follicular fluid protein in follicles of human ovaries.

Human ovarian follicular fluid protein has been partially purified and the active fraction designated as hGF2. Using specific polyclonal antiserum to hGF2, it was observed to be localized immunohistochemically in the granulosa cells of medium but not large follicles of human ovary. The hGF2 levels were estimated by ELISA in serum and follicular fluid of 10 gonadotropin-stimulated women recruited for IVF-ET programme. The results revealed a 3-fold increase in the concentration of hGF2 in follicular fluid compared to that in serum of these patients. These data indicate that the protein is secreted by granulosa cells and plays an important role in the regulation of follicular maturation and ovulation.