A New Marker for the Diagnosis of Acute Pelvic Inflammatory Disease: Immature Granulocyte.

BACKGROUND: The goal is to assess the role of immature granulocytes (IG) in the diagnosis of acute pelvic-inflammatory-disease (PID) and to determine whether they are useful for discriminating mild/moderate and severe PID. METHODS: Patients admitted with the diagnosis of acute PID were retrospectively assessed. Diagnosis was based on CDC criteria. Patients were grouped as severe and mild/moderate PID based on need for hospitalization. Control group consisted of patients in whom PID was excluded by laparoscopy. Sample size was calculated with statistical methods. IGs were compared within the groups. Cutoff values were determined for prediction of diagnosis and severity of acute PID. RESULTS: There were 74 severe, 32 mild/moderate acute PID, and 41 control patients. Thirty patients had surgery following no response to antibiotic treatment or tubo-ovarian abscess. IGs were significantly higher in the severe group compared to mild/moderate and control groups. ROC analysis showed IG counts (≥ 0.035 µL) and percentages (≥ 0.35%) were significantly effective in predicting acute PID and were associated with severity when they were ≥ 0.055 µL and ≥ 0.42%, respectively. IG count ≥ 0.085 was found to have 58.6% sensitivity and 63.1% speci-ficity for prediction of surgical intervention need. CONCLUSIONS: IGs are components of simple CBC tests and are easily obtainable, cheap markers. They were found to be elevated in acute PID and correlated significantly with the severity of the disease. These markers may serve as adjunctive markers for the diagnosis of acute PID and may be useful in discrimination between mild/moderate and severe PID.

Molecular characterization of acutely and gradually heavy metal acclimated aquatic bacteria by FTIR spectraoscopy.

In the environment, bacteria can be exposed to the concentration gradient of toxic heavy metals (gradual) or sudden high concentration of them (acute). In both situations, bacteria get acclimated to toxic heavy metal concentrations. Acclimation causes metabolic and molecular changes in bacteria. In this study, we aimed to understand whether there are differences between molecular profiles of the bacteria (Brevundimonas, Gordonia and Microbacterium) which are under acute or gradual exposure to cadmium or lead by using ATR-FTIR spectroscopy. Our results revealed the differences between the acclimation groups in membrane dynamics including changes in the structure and composition of the membrane lipids and proteins. Furthermore, protein concentrations decreased in acclimated bacterial groups. Also, a remarkable increase in exopolymer production occurred in acclimated groups. Interestingly, bacteria under acute cadmium exposure produced the significantly higher amount of exopolymer than they did under gradual exposure. On the contrary, under lead exposure gradually acclimate strains produced significantly higher amounts of exopolymer than those of acutely acclimated ones. This information can be used in bioremediation studies to obtain bacterial strains producing a higher amount of exopolymer.

Mycobacterium avium Subsp. paratuberculosis Induces Specific IgE Production in Japanese People with Allergies.

Background. The prevalence of allergies is steadily increasing worldwide; however, the pathogenesis is still unclear. We hypothesized that Mycobacterium avium subsp. paratuberculosis (MAP) may contribute to allergy development. This organism can be present in dairy foods, it can elicit an immunomodulatory switch from a Th1 to a Th2 response, and it has been speculated that it is linked to several human autoimmune diseases. To determine the contribution, sera from 99 individuals with various atopic disorders and 45 healthy nonallergic controls were assessed for total IgE levels and successively for MAP-specific IgE by ELISA. Results. The mean total serum IgE level in allergic patients was 256 ± 235 IU/mL, and in the healthy controls it was 62 ± 44 IU/mL (AUC = 0.88; p < 0.0001). Among the patient groups, 50 of the 99 subjects had increased IgE total level ≥ 150 IU/mL, while 49 subjects had IgE ≤ 150 IU/mL (mean level: 407 ± 256 IU/mL versus 106 ± 16 IU/mL; p < 0.0001). Additionally, 6 out of 50 subjects (12%) with IgE ≥ 150 IU/mL and none (0%) with IgE ≤ 150 IU/mL were positive for specific MAP IgE (AUC = 0.63; p = 0.03). Conclusion. The present study revealed that MAP has the ability to induce specific IgE and might contribute to the induction of allergic inflammation in genetically predisposed individuals.

Three-dimensional evaluation of subclinical extension of extramammary Paget disease: visualization of the histological border and its comparison to the clinical border.

BACKGROUND: In extramammary Paget disease (EMPD), Paget cells are sometimes detected outside the clinical border (subclinical extension). However, the spreading pattern of Paget cells in subclinical extension remains unclear. In addition, the macroscopic appearances of lesions accompanied by subclinical extension are totally unknown. OBJECTIVES: To characterize the spreading pattern of Paget cells as well as the macroscopic appearance of lesions of EMPD with subclinical extension. METHODS: Nineteen patients with primary anogenital EMPD underwent mapping biopsies and excisional surgeries; biopsy samples were then taken at the periphery of well-demarcated lesions. Samples were transparentized and subjected to whole-mount immunostaining with anticytokeratin 7 antibody to label Paget cells. The histological border was evaluated in three dimensions by two-photon microscopy. The shape and location of the histological border were compared with those of the clinical border. RESULTS: In 21 samples taken at the lesion where subclinical extension was not shown by mapping biopsy, the shape and location of the histological border were almost identical to those of the clinical border. However, two samples exhibited small foci of Paget cells outside the clinical border, showing subclinically extended satellite lesions. In the two samples taken at the lesions where subclinical extension was shown by mapping biopsy, a continuous arrangement of Paget cells extending beyond the clinical border was identified. Subclinically extended Paget cells were detected solely outside hypopigmented patches with erythema. CONCLUSIONS: In EMPD, at least two patterns of subclinical extension exist: continuous and satellite lesions. Subclinical extension might exist preferentially outside hypopigmented patches with erythema.

Perturbations of both nonregulatory and regulatory FOXP3+ T cells in patients with malignant melanoma.

BACKGROUND: 'FOXP3+ regulatory T cells' (Tregs) are reported to be increased in tumour-bearing hosts including patients with melanoma, leading to tumour immune suppression. However, this idea is challenged by recent evidence that the 'FOXP3+ Treg' fraction in fact contains activated 'nonregulatory' T cells. Also, FOXP3+ T cells are reported to have functionally and kinetically distinct subsets. OBJECTIVES: To investigate whether either or both of regulatory and 'nonregulatory' FOXP3+ T cells are perturbed in patients with melanoma. METHODS: FOXP3+ T cells were classified into three subsets, namely CD45RO+FOXP3(low) nonregulatory T cells, CD45RO+FOXP3(high) effector Tregs, and CD45RO-FOXP3(low) naïve Tregs, according to their expression levels of FOXP3 and CD45RO. The percentage and cytokine production of these FOXP3+ T-cell subsets were assessed by flow cytometry. RESULTS: Both regulatory and nonregulatory T cells were increased in patients with melanoma. Moreover, we found three unexpected perturbations in FOXP3+ T-cell subsets: (i) patients with melanoma showed higher frequencies of FOXP3(low) nonregulatory T cells, which decreased and normalized after tumour removal; (ii) FOXP3(low) naïve Tregs containing higher frequencies of interferon-γ+ cells increased with tumour progression; and (iii) CD45RO+FOXP3(high) effector Tregs were pronouncedly infiltrated around tumour tissues. CONCLUSIONS: These findings demonstrate that patients with melanoma have distinct and differential perturbation of both regulatory and nonregulatory FOXP3+ T cells. The degree of perturbation is associated with tumour burden and progression, suggesting that the perturbation reflects fundamental pathophysiological processes in patients with melanoma. The presented analysis provides a practical approach to investigate the immunological environment of cancer patients.

Monitoring stem cell proliferation and differentiation in primary midgut cell cultures from Heliothis virescens larvae using flow cytometry.

In the midgut of Heliothis virescens larvae, proliferation and differentiation of stem cell populations allow for midgut growth and regeneration. Basic epithelial regenerative function can be assessed in vitro by purifying these two cell type populations, yet efficient high throughput methods to monitor midgut stem cell proliferation and differentiation are not available. We describe a flow cytometry method to differentiate stem from mature midgut cells and use it to monitor proliferation, differentiation and death in primary midgut stem cell cultures from H. virescens larvae. Our method is based on differential light scattering and vital stain fluorescence properties to distinguish between stem and mature midgut cells. Using this method, we monitored proliferation and differentiation of H. virescens midgut cells cultured in the presence of fetal bovine serum (FBS) or AlbuMAX II. Supplementation with FBS resulted in increased stem cell differentiation after 5 days of culture, while AlbuMAX II-supplemented medium promoted stem cell proliferation. These data demonstrate utility of our flow cytometry method for studying stem cell-based epithelial regeneration, and indicate that AlbuMAX II-supplemented medium may be used to maintain pluripotency in primary midgut stem cell cultures.

Malignant melanoma derived from cerebriform intradermal naevus.

We report a case of malignant melanoma (MM) derived from cerebriform intradermal naevus (CIN) in a 66-year-old Japanese man. The patient had cutis verticis gyrata (CVG) on the posterior area of the scalp at birth. He noticed a dome-shaped nodule at the centre of the CVG at 66 years of age. Histopathological examination found a nodule of MM arising within an extensive area of intradermal naevus. There was no metastasis to lymph nodes or other organs. To our knowledge, only two cases of CIN in which MM had later developed have been reported. We estimated that the incidence of melanoma from CIN including our case is 4.5% (3 of 67 reported cases), which seems to be comparable to the frequency of malignant alteration of giant pigmented naevi. This suggests that pathological examination is recommended for CVG, and once pathological diagnosis of CIN is confirmed, long clinical follow-ups are necessary for detecting development of MM.

Extensive skin necrosis of the arm in a patient with complex regional pain syndrome.

We report a 36-year-old woman with complex regional pain syndrome (CRPS) type 1 presenting with extensive skin necrosis of the left arm. The patient cooled her arm with ice packs to ease severe pain due to CRPS, in spite of repeated cautions against frostbite injury. The regions of skin necrosis corresponded with the sites where she had applied ice packs. We considered that the severe skin necrosis in our case was due to a self-induced frostbite injury.

A case of atypical sporotrichosis with multifocal cutaneous ulcers.

We report an atypical case of sporotrichosis in an elderly woman working as a horticulturist, who presented with multiple ulcers and nodules on the face and the right upper back. Histological examination found numerous small yeast-like spores in the granulomatous reaction in the upper dermis. Culture and DNA analysis identified Sporothrix schenckii, group B. Misuse of topical steroids and self-inoculation may have caused the atypical features found in this patient.

Estrogen recruits the endocannabinoid system to modulate emotionality.

Estrogen administration elicits anxiolytic and antidepressant-like effects in female rats; however, the mechanism of this effect is unknown. Fatty acid amide hydrolase (FAAH), the enzyme which degrades the endocannabinoid anandamide, has been shown to be regulated by estrogen. Thus, we examined if the anxiolytic and antidepressant effects of estrogen implicated the endocannabinoid system. In the first experiment, ovariectomized female rats were administered a single injection of 17beta-estradiol (10 microg) or oil, and 48 h later were given an injection of the cannabinoid CB1 receptor antagonist AM251 (1 mg/kg) or vehicle. One hour after AM251 or vehicle administration, subjects were tested in either the open field test (OFT), elevated plus maze (EPM) or the forced swim test (FST). Estradiol treatment resulted in a significant increase in open arm entries in the EPM and time spent in the center quadrant of the OFT, which were reversed by co-treatment with AM251, suggesting that endocannabinoids are integral to the anxiolytic effects of estrogen. No significant effects of estradiol or AM251 were seen in the FST. In the second experiment, administration of the FAAH inhibitor URB597 (0.1 and 0.3 mg/kg) increased open arm entries in the EPM and time spent in the center quadrant in the OFT as well as significantly reduced immobility in the FST. Collectively, these data demonstrate that estrogen may elicit changes in emotional behavior through an endocannabinoid mechanism, and suggest that inhibition of FAAH represents a therapeutic target for anxiety and depression in women.

New method of serological testing for Mycobacterium avium subsp. paratuberculosis (Johne's disease) by flow cytometry.

Johne's disease (JD) or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Attempts to control JD have proven inordinately difficult due to low levels of sensitivity by currently available diagnostic tests, which are also incapable of detecting prepatent MAP infections. In the present work, we describe the use of a flow cytometry method (FCM) for serological diagnosis of subclinical and clinical JD in cattle. The FCM was capable of distinguishing MAP-infected from MAP-non-infected cattle as well as MAP from M. scrofulaceum and M. avium subsp. avium. Results of the FCM were compared to that of a commercially available ELISA using 82 serum samples from JD-positive and JD-negative dairy and beef cattle farms that were separated into the following groups: (1) sera from a JD-free farm; (2) sera from JD-positive farms that had tested negative by ELISA; and (3) sera from JD-positive farms that tested JD-positive by ELISA. The FCM found that groups 1-3 were 6.6%, 73.3%, and 97.3% positive for MAP infections, respectively. By using 30 fecal culture-negative samples from a JD-free farm and 21 fecal culture-positive samples from JD-positive farms, diagnostic sensitivity and specificity of the FCM were calculated to be 95.2% and 96.7%, respectively. A retrospective study of 10 JD-positive cows showed that the FCM detected MAP infections 6-44 months earlier than the fecal culture test. Further, the FCM specifically detected MAP infections in serum samples as early as 170 days after experimental inoculation of calves with MAP and did not react with calves inoculated with other mycobacteria. Production of IgG against MAP was detected by FCM in all the calves inoculated with MAP 240 days after inoculation, whereas positive anti-MAP IgG production was not detected in control calves or calves experimentally infected with M. avium subsp. avium or M. bovis. The FCM assay is rapid and is completed in less than 4 h. Moreover, the FCM is objective, technically easy and can be automated for handling large numbers of samples. This novel assay might form the basis of a highly sensitive and subspecies-specific test for the diagnosis of JD.

Selection of peptides recognized by human antibodies against the surface of Plasmodium falciparum-infected erythrocytes.

In an attempt to identify mimotopes of the surface antigens of P. falciparum-infected erythrocytes (iRBC), antibodies were eluted from iRBC that had been treated with a pool of sera from malaria-infected individuals (IHS), and were used to screen a phage display library (PDL). After repeated panning of the PDL on immobilized antibodies, phage that selectively bound to IHS were accumulated. Of 23 randomly chosen clones that were sequenced, 13 individual sequences were detected at varying frequencies and 3 of the 13 sequences had homology with membrane proteins known to exist on iRBC. The majority of phage clones (7 out of 8 clones) selected after the 4th panning bound selectively to IgG in IHS. Specific binding of the selected phage to IgG in IHS was also confirmed using 24 IHS and 11 sera from uninfected individuals. One phage clone was the most frequently found in the sequenced clones after the 4th panning, and the binding of this clone to IgG in all IHS was greater than in any serum from uninfected individuals. A rabbit antiserum against the peptide expressed on the clone specifically recognized the surface of iRBC and resulted in iRBC haemolysis.

Erythrocyte aging and malaria.

The human malaria parasite, Plasmodium falciparum, ages the red blood cell during its intracellular development. During this process of erythrocyte senescence the parasitized cell becomes less dense and deformable, its biconcave disc shape becomes more spherical and is covered with microscopic protuberances (knobs); the amounts of membrane cholesterol and phospholipids are altered and phosphatidylserine (PS) is externalized. The malaria-infected cell is osmotically fragile, more permeable to a wide variety of molecules via new permeation pathways (NPP), and there is surface deposition of immunoglobulins and complement. There are declines in sialic acid, reduced glutathione, tocopherol and ATP. Hemichromes are deposited on the inner surface of the red cell membrane and there is clustering of the anion transporter, band 3 protein, as well as exposure of neoantigens which contribute to antigenic variation and adhesivity of the parasitized erythrocyte. These time-dependent changes result from oxidative assault and a combination of factors, including a decline in levels of anti-oxidants and ATP coupled with an enhanced flux of ions especially calcium. Despite these parasite-induced age effects P. falciparum is able to avoid destruction by splenic removal through microvessel sequestration in the deep tissues via PS, clustered band 3 protein and adhesive neoantigens.

Cell cycle analysis and expression of cell cycle regulator genes in myeloma cells overexpressing cyclin D1.

Among the recently discovered myeloma-specific gene alterations associated with chromosomal translocations, cyclin D1/PRAD1/Bcl-1 overexpression caused by t(11;14)(q13;q32) is considered to be the most frequent in myeloma patients and cell lines, and may be a prognostic factor clinically. To elucidate the cellular biological role of overexpressed cyclin D1 in myeloma cells, we examined the mRNA expression levels of cell cycle regulators including three cyclin Ds, cyclin-dependent kinase inhibitors (CDK-Is) and accelerators. Cyclin D1 overexpression was clearly demonstrated in the lines with abnormal 11q13 and associated with overexpression of S and G2 accelerator genes. The cyclin D1-overexpressing lines tended to have a shortened G1 phase compared with the non-expressing lines. In addition, artificial silencing using antisense oligonucleotides for cyclin D1 suppressed the growth rate of some but not all cyclin D1-overexpressing cells. These results indicate that overexpression of cyclin D1 caused by cytogenetic abnormalities may make cells progress through the cell cycle rapidly, but it seems that other factors such as cyclin D2 and translocation-related genes affect the cell cycle progression in myeloma cells.