Laboratorial aspects of cytolytic vaginosis and vulvovaginal candidiasis as a key for accurate diagnosis: a pilot study / Aspectos laboratoriais da vaginose citolítica e candidíase vulvovaginal como uma chave para o diagnóstico preciso: Um estudo piloto

Abstract Objective To identify clinical, microscopic, and biochemical characteristics that differentiate cytolytic vaginosis (CV) from vulvovaginal candidiasis (VVC). Methods The present cross-sectional study analyzed the vaginal contents of 24 non-pregnant women aged 18 to 42 years who were attended at the Genital Infections Clinic at Centro de Atenção Integral à Saúde da Mulher da Universidade Estadual de Campinas (CAISM-UNICAMP). They were diagnosed either with (CV = 8, VVC = 8) or without vulvovaginitis or vaginal dysbiosis (controls). The socio-demographic, clinical, and gynecological data were obtained from a detailed patient interview. Samples of the vaginal contents were collected for analysis of vaginal pH, gram stain, and specific fungal culture. The Kruskal-Wallis and Fisher exact tests were used to compare the differences between the groups. Odds ratios were used to compare the categorical variables. The significance level was considered at p < 0.05. Results Both women with CV and VVC had a lumpy vaginal discharge (p = 0,002) and vaginal hyperemia (p = 0.001), compared with controls. The inflammatory process was more intense in the VVC group (p = 0.001). In the CV group, there was statistical significance for the lactobacillus amount (p = 0.006), vaginal epithelium lysis (p = 0.001), and vaginal pH (p = 0.0002). Conclusion Cytolytic vaginosis and VVC diagnoses rarely differ on clinical characteristics but have different laboratorial findings. The present study highlights the importance of conducting an accurate investigation through laboratory tests rather than clinical criteria to avoid misdiagnosis.

Resumo Objetivo Identificar características clínicas, microscópicas e bioquímicas que diferenciam a vaginose citolítica (VC) da candidíase vulvovaginal (CVV). Métodos O presente estudo de corte transversal analisou o conteúdo vaginal de 24 mulheres não grávidas, com idades entre 18 e 42 anos, atendidas no ambulatório de Infecções Genitais do Centro de Atenção Integral à Saúde da Mulher da Universidade Estadual de Campinas (CAISM-UNICAMP). Elas foram diagnosticadas com (CV = 8, CVV = 8) ou sem vulvovaginite ou disbiose vaginal (controles = 8). Os dados sociodemográficos, clínicos e ginecológicos foram obtidos em uma entrevista detalhada do paciente. Amostras do conteúdo vaginal foram coletadas para análise do pH vaginal, coloração de Gram e cultura específica de fungos. Os testes exatos de Kruskal-Wallis e Fisher foram utilizados para comparar as diferenças entre os grupos. A razão de chances foi utilizada para comparar as variáveis categóricas. O nível de significância considerado foi de p < 0,05. Resultados As mulheres com VC e CVV apresentaram corrimento vaginal irregular (p = 0,002) e hiperemia vaginal (p = 0,001), em comparação aos controles. O processo inflamatório foi mais intenso no grupo CVV (p = 0,001). No grupo VC, houve significância estatística para a quantidade de lactobacilos (p = 0,006), lise do epitélio vaginal (p = 0,001) e pH vaginal (p = 0,0002). Conclusão Os diagnósticos de VC e CVV raramente diferem nas características clínicas, mas apresentam achados laboratoriais diferentes. O presente estudo destaca a importância de conduzir uma investigação precisa por meio de testes laboratoriais, em vez de critérios apenas clínicos, a fim de evitar erros de diagnóstico.

Vaginitis citolítica y los errores terapéuticos: Cómo hacer un diagnóstico y tratamiento diferenciado de la vaginitis candidiásica recurrente / Cytolystic vaginitis. How to make a diagnosis and differentiated treatment from recurrent caginitis vaginitis

Introducción: La vaginitis citolítica (VC) se refiere a la lisis de las células del epitelio intermedio de la vagina causado por un aumento de lactobacilos que provocan acidificación extrema de la vagina. Los síntomas pueden llevar a confundir su diagnóstico con vulvovaginitis candidiásica (VVC), por lo que puede ser tratada de manera errónea con antimicóticos sin responder al tratamiento. Objetivo: Realizar una revisión bibliográfica sobre el diagnóstico y tratamiento diferencial de la VC. Métodos: Se realizó una búsqueda bibliográfica en varias bases de datos: Academic Google, Scopus, PubMed, LILACS, ClinicalKey utilizando los términos MeSH y DeCS: "vaginitis citolítica", "cytolitic vaginitis", "vulvovaginitis candidiásica", "vaginitis recurrente". Resultados: Se identificaron 42 artículos de los cuales se seleccionó 34 para la presente revisión. Discusión: En la VC a través de estudios complementarios se puede identificar abundantes lactobacilos, especialmente L. crispatus y pH vaginal inferior a lo normal. El examen lipídico de secreciones vaginales, sugiere la existencia de niveles elevados de lípidos relacionados con apoptosis celular, estrés oxidativo y sobrecrecimiento bacteriano. El objetivo del tratamiento es mejorar la exagerada acidez vaginal para lo cual se recomienda el empleo de baños de asiento o duchas vaginales con bicarbonato de sodio entre otras medidas. Conclusiones: La VC es una entidad frecuente pero poco conocida, comúnmente confundida con VVC. Su tratamiento debe evitar el uso de antimicóticos.

Oral Administration of Silk Peptide Enhances the Maturation and Cytolytic Activity of Natural Killer Cells

Silk peptide, the hydrolysate of silk protein derived from cocoons, has been employed as a biomedical material and is believed to be safe for human use. Silk peptide display various bioactivities, including anti-inflammatory, immune-regulatory, anti-tumor, anti-viral, and anti-bacterial. Although earlier investigations demonstrated that silk peptide stimulates macrophages and the production of pro-inflammatory cytokines, its effect on natural killer (NK) cell function has not yet been explored. In this study, we initially confirmed that silk peptide enhances NK cell activity in vitro and ex vivo. To assess the modulatory activity of silk peptide on NK cells, mice were fed various amounts of a silk peptide-supplemented diet for 2 months and the effects on immune stimulation, including NK cell activation, were evaluated. Oral administration of silk peptide significantly enhanced the proliferation of mitogen- or IL-2-stimulated splenocytes. In addition, oral silk peptide treatment enhanced the frequency and degree of maturation of NK cells in splenocytes. The same treatment also significantly enhanced the target cell cytolytic activity of NK cells, which was determined by cell surface CD107a expression and intracellular interferon-γ expression. Finally, oral administration of silk peptide stimulated T helper 1-type cytokine expression from splenic lymphocytes. Collectively, our results suggest that silk peptide potentiates NK cell activity in vivo and could be used as a compound for immune-modulating anti-tumor treatment.

Vaginosis citolítica en pacientes con diagnóstico clínico de vulvovaginitis candidiásica / Cytolytic vaginosis in patients with clinical diagnosis of vulvovaginal candidiasis

La microbiota vaginal en la edad fértil está dominada por Lactobacillus spp. Su sobrecrecimiento provoca el aumento en la producción de ácido láctico, disminución del pH vaginal y lisis de células del epitelio vaginal, causando vaginosis citolítica, la cual se confunde con vulvovaginitis candidiásica (VVC) por la aparición de signos y síntomas similares. Se determinó la prevalencia de vaginosis citolítica, así como sus características clínicas y epidemiológicas en pacientes que consultaron con clínica sugestiva de VVC durante el período agosto-septiembre del año 2015. Se encontraron 12 pacientes con clínica compatible de VVC. En 11 (91,7%) se realizó el diagnóstico de vaginosis citolítica y en 1 (8,3%) de vulvovaginitis candidiásica. El 63,7% de las pacientes (7) tenían diagnóstico previo de VVC recurrente. Los síntomas más frecuentes fueron prurito vaginal o vulvar y secreción vaginal blanquecina de aspecto grumoso como leche cortada. La clínica iniciaba entre los 4-6 días posteriores a la menstruación, y desaparecía días antes o justo con el inicio de una nueva fase menstrual. En conclusión, se encontró que en las pacientes examinadas la sintomatología de ambas patologías resultó indistinguible, por tanto el diagnóstico clínico no es suficiente y debe recurrirse al diagnóstico de laboratorio para distinguir entre vaginosis citolítica y vulvovaginitis candidiásica.

The vaginal microbiota in fertile age is dominated by Lactobacillus spp. Its overgrowth causes increased production of lactic acid, decreased vaginal pH and lysis of cells of the vaginal epithelium, causing cytolytic vaginosis, which can be confused with vulvovaginal candidiasis (VVC) due to the similar signs and symptoms. The prevalence of cytolytic vaginosis, as well as its clinical and epidemiological characteristics was determined in patients who consulted with suggestive signs and symptoms of VVC during the period August to September of the year 2015. Twelve patients with signs and symptoms compatible with VVC were considered. Of those, 11 (91.7%) were diagnosed as cytolytic vaginosis and 1 (8.3%) corresponded to vulvovaginal candidiasis. From the 12 patients included, 7 (63.7%) had previous diagnosis of recurrent VVC. The most frequent symptoms recorded were vaginal or vulvar pruritus and whitish, cheesy or curd-like vaginal discharge. Signs and symptoms started between 4 to 6 days after menstruation, and disappeared days before or just with the onset of a new menstrual cycle. In conclusion, it was found that in the patients examined, signs and symptoms of both pathologies were indistinguishable. Therefore, the clinical diagnosis is not enough and laboratory studies should be used to distinguish between cytolytic vaginosis and vulvovaginal candidiasis.

Vaginosis citolítica: presentación de un caso clínico / Cytolytic vaginosis: presentation of a clinical case

La vaginosis citolítica, descrita hace 30 años como citólisis de Döderlein, es frecuente en mujeres en la edad reproductiva y, por las características de flujo vaginal blanquecino y síntomas clínicos, es indistinguible de la vulvovaginitis micótica. Se presenta el caso de una paciente, con diagnóstico clínico presuntivo de vulvovaginitis por Candida spp. a repetición, tratada empíricamente con antifúngicos por año y medio sin ninguna mejoría. Luego de estudios microbiológicos, la coloración de Gram, demostró la presencia de 50 bacilos grampositivos por campo y abundantes núcleos celulares desnudos. El cultivo resultó puro para Lactobacillus spp., lo que permitió confirmar el diagnóstico de vaginosis citolítica. La paciente fue tratada con ampicilina-sulbactam y no ha vuelto a presentar recidivas. En conclusión, es fundamental determinar el pH vaginal de las pacientes en la consulta, así como practicar una coloración de Gram de la secreción vaginal para poner en evidencia los cambios celulares por el exceso de ácido en la vagina y así evitar tratamientos antifúngicos innecesarios que acrecentarán los trastornos de la microbiota vaginal.

Cytolytic vaginosis, described 30 years ago as Döderlein cytolysis, is common in women of reproductive age and, due to the characteristics of whitish vaginal discharge and clinical symptoms, is indistinguishable from mycotic vulvovaginitis. We describe the case of a patient with presumptive clinical diagnosis of recurrent vulvovaginitis by Candida spp. treated empirically with antifungal agents for one and a half years without improvement. After microbiological studies, Gram staining demonstrated the presence of 50 Gram-positive bacilli per field and abundant nude cell nuclei. The culture recovered pure Lactobacillus spp. which permitted the diagnosis of cytolytic vaginosis. The patient was treated with ampicillin-sulbactam and since, has not had recurrences. In conclusion, it is essential to examine the pH of the patient vaginal discharge, as well as to practice a Gram staining of the vaginal secretion to demonstrate the cellular changes produced by the excess of acid in the vagina and therefore avoid unnecessary antifungal treatments that will produce undue changes of the vaginal microbiota.

The effects of JNK1 on the responses of cytolytic T cells against viral infection / 中华微生物学和免疫学杂志

Objective To examine the role of c-Jun NH2-terminal kinase(JNK) 1 in cytolytic T lymphocyte (CTL) responses against virus infection.Methods Wild-type (WT) and JNK1-knockout (JNK1-/-) mice were infected with ectromelia virus(ECTV) through hind footpads.Survival and virus titers in the target organs (liver and spleen) were analyzed.Effector T cells in the spleen and popliteal lymph nodes were determined on day 3 and 7 post-infection.Proliferation and INF-γ production of CTL were also detected.Results JNK1 deficiency caused an increased susceptibility to ECTV infection in mice,indicated by higher case fatality and viral burden in target organs.The decrease in CTL response correlated with a defect in CTL proliferation and INF-γproduction.Conclusion The data suggest that JNK1 is involved in expansion of activated CTL during ECTV infection,and plays an important antiviral role in regulating the proliferation and effector function of CTL.

Biological activity of sea anemone proteins: II. Cytolysis and cell line toxicity.

Potent cytolytic activity was exhibited by proteins extracted from three sea anemones viz. Heteractis magnifica, Stichodactyla haddoni and Paracodylactis sinensis by affecting the red blood corpuscles (RBC) and the mouse fibroblast cell line (L929) and leukemia cell line (P388). Crude toxin of all the three anemone species induced spontaneous hemolysis of chicken, goat and human erythrocytes. The crude toxin of H. magnifica (0.98 mg/ml) elicited hemolysis at levels of 4096, 512 and 4096 HU (hemolytic unit) in chicken, goat and human erythrocytes respectively. Subsequently, the crude toxin of S.haddoni (0.82 mg/ml) exhibited a hemolytic activity of 256, 128 and 512 HU and that of P. sinensis (0.60 mg/ml) had a hemolytic activity of 128, 4096 and 512 HU. Most of the partially purified proteins of these anemones also exhibited the activity against the three different erythrocytes. The viability of L929 and P388 was adversely affected on adding the crude toxins. The symptoms of toxicity shown by the cells were rounding, lysis and detachment from the substratum. These effects were the least in S. haddoni, as compared to those the crude toxins of the other two species. Inhibition of growth of L929 exhibited by the toxin of the three species ranged between 61.08 and 93.38%. Similarly, inhibition of the growth of P388 ranged between 51.32 and 86.16%. The present investigation reveal the cytotoxic nature of anemone toxins.

Vaginosis citolítica: un diagnóstico diferencial poco frecuente de vulvovaginitis micótica a repetición / Cytolytic vaginosis: a rare differential diagnosis of fungal recurrent vulvovaginitis

Los síntomas compatibles con vaginitis y vaginosis, son la causa más frecuente de consulta al ginecólogo. La descarga vaginal abundante y el prurito, constituyen la primera sospecha de estas patologías. Sin embargo, debemos tener presente que el aumento significativo de lactobacilos, conocido como lactobacilosis, puede expresarse también con los mismos síntomas y signos de una vulvovaginitis micótica. Si esta condición se mantiene en el tiempo, puede provocar un grado de acidez anormal en la vagina, y puede llevar a una lisis de las células epiteliales que se conoce como vaginosis citolítica. Este diagnóstico, aunque es poco frecuente, debemos sospecharlo especialmente, en aquellas mujeres que relatan tener múltiples consultas por candidiasis vaginal, con escasa o nula respuesta frente a la terapia antimicótica.

The symptoms consistent with vaginitis and vaginosis are the most common cause of the visit to the gynecologist. Heavy vaginal discharge and pruritus are the first suspicion of this pathology. However, the significant increase in lactobacilli, known as lactobacilosis can be expressed also with the same symptoms and signs of a fungal vulvovaginitis. If this condition is maintained overtime, can cause abnormal acidity in the vagina, and may lead to epithelial cell lysis, which is known as cytolytic vaginosis. This diagnosis, although rare, should be suspected especially in women who reported having multiple consultations for vaginal candidiasis, with little or no response to antifungal therapy.

Genetic construction of HLA-A2 restricted multi-epitopes gene of hepatitis C virus,expression and purification in E.coli for antigenic analysis / 中国免疫学杂志

Objective:To construct the recombinant prokaryotic plasmid to express HCV HLA-A2 restricted multi-CTL epitopes and to purify the fused protein for antigenic analysis.Methods:The human ubiquitin gene and multi-CTL epitopes gene was synthesized respectively,and digested by restrict enzyme before being cloned into pRSET-A.Then it was transformed into E.coli DH5α and the positive recombinant plasmid named pRSET-Ub-Mep was sequenced.Target protein was distinctly expressed after transformed into E.coli BL21 and induced with IPTG.Thus the protein was scanned and purified on Ni~(2+)-NTA column as well as Western blot performed after solubility analysis.Results:The recombinant plasmid pRSET-Ub-Mep was successfully constructed and it could efficiently express the target gene.Protein production was mainly in inclusion body and could be purified through Ni~(2+)-NTA column.The purified protein kept the antigen activity.Conclusion:The gene encoding for HCV HLA-A2-restricted multi-CTL epitopes is efficiently expressed and the target protein is purified,which establishes a foundation of further research to evaluate the cellular immune response induced by the target gene.

Cytolytic Activity Analysis of Grass Carp Perforin C-terminal Peptide / 中国生物工程杂志

The cytolytic effect of perforin is a mechanism of anti-virus,killing microbial-infected cells and tumor cells.Perforin is a very important non-specific immune factors in fish.In order to understand the function of perforin,the cDNA of grass carp perforin C-terminal peptide was amplified from grass carp liver and kidney cDNA library.It contains a protein kinase C conserved region 2(C2).The cDNA was connected with pET32a,and transformed to expression bacteria DE3.PFP-C was expressed by a prokaryotic expression system and then purified by affinity chromatography.It showed a significant haemolytic activity when tested with rabbit red cells,the optimal pH for haemolytic activity was 7.5,and its haemolytic function dependents on Ca2+ apparently.

Role of adenovirus mediated CTLA4-Ig gene in the immune cells infiltration and cell apoptosis in murine liver transplantation / 中国普通外科杂志

Objective To investigate the role of B7/CD28 costimulation pathway blockade with adenovirus-mediated CTLA4-Ig gene in macrophage and CD8~+T cell infiltration and cell apoptosis in murine liver transplantation. Methods Rat pairs were divided into three groups: SD-to-Wistar transplantation control group, CsA-treated group and CTLA4-Ig-treated group. IHC and TUNEL were used to analyze the expression of CTLA4-Ig gene in liver and immune cells infiltrate and cell apoptosis in liver grafts. Pathology was done on all harvested grafts. ResultsCTLA4-Ig gene expression was positive in the donor liver on day 7 after administering adenovirus-mediated CTLA4-Ig gene via vein, and remained positive until day 60 after liver transplantation. Infiltration of immune cells in CTLA4-Ig-treated group was less than that in rejection control group. the apoptotic index of rejection group on day 3,5,7 was significantly higher than those of CTLA4-Ig-treated. Conclusions CTLA4-Ig gene was constantly expressed in the donor liver after single intravenousely injection into rats using adenovirus as vector. Adenovirus-mediated CTLA4-Ig gene therapy can inhibit infiltration of immune cells and apoptosis in grafts, thus prolonging the survival of recipients.

Induction of specific cytotoxic T lymphocytes using hepatoma antigenic peptide mixed with HSP70 in vitro / 中国免疫学杂志

Objective:To explore the possibility of inducing cell mediated immune response with HSP70 antigenic peptide complex in vitro.Methods:HSP70 peptide complex was reconstituted in vitro.Granulocyte/macrophage colony stimulating factors and interleukin 4 were used to cultivate DC from peripheral blood of HLA A2 positive healthy donors.HSP70,HSP70 peptide complex or peptide was used to activate the DC individually,which will initiate homogenize T lymphocyte to form cytotoxic T lymphocyte(CTL).The cytotxicity of the CTL was detected by MTT assay.Results:It was found that peptide specific CD8 + CTL responses were readily elicited by HSP70 peptide complex or peptide.The CTL response primed by HSP70 peptide complex was more potene than peptide alone.Conclusion:The results suggest that HSP70 peptide complex is immunogenic and HSP70 can lead to great efficient CTL response,antigenic peptides and HSP70 complex may be used as peptide vaccines for cancer immunotherapy.

Effects of Panax ginseng Aqua - acupuncture on Lymphocyte Activities in Glucocorticoid Treated Mice / 대한면역학회지

Panax ginseng (PG) has been used as an important analeptic in traditional medicine. This study was purposed to investigate the effect of PG on immune responses induced by glucocorticoid in mice. PG solution was injected into CV6 and BL23, which are the classical acupuncture points, for 7 days after injection with glucocorticoid. And then B and T cell proliferation and cytolytic activity of natural killer (NK) cells were measured. B cell proliferation by 'H-thymidine incorporation was decreased by about 25% in control group as compared with normal group. However, B cell proliferation was significantly increased 1.8-fold in CV6 group and 2.5-fold in BL23 group as compared with normal group. T cell proliferation by H- thymidine incorporation was decreased by about 15% in control group as compared with normal group. On the other hand, T cell proliferation was significantly increased 1.9-fold in CV6 group and 2.3-fold in BL23 group as cornpared with normal group. Furthermore in purified T cell, the proliferation was furtherly increased rather than in non-purified T cell. The activity of NK cell was remarkably decreased in control group as compared with normal group. However, the activities of NK cells in CV6 and BL23 groups were recovered to the above levels of normal group. On the other hand, the activity of NK cell in the blank locus group was slightly increased compared with control group. However this increasement was not reached the levels of CV6 and BL23 groups. And in the case of purified NK cell, the cytolytic activity of NK cell was respectively increased 1.6-fold in normal group, 1.4-fold in control group, 2.0-fold in blank locus group and 2.0-fold in CV6 group and 1.4-fold in BL23 group as compared to the non-purifed NK cell. These results suggest that PG aqua-acupuncture at CV6 and BL23 may proliferate B and T cells that is suppressed by glucocorticoid, and activate NK cell activity.

Effect of perforin-mediated cytotoxicity on systemic defense mechanisms against primary influenza virus infection / 中国免疫学杂志

Objective:To investigate the effect of perforin-mediated cytotoxicity in primary influenza virus infection.Methods:Perforin-deficient and wild-type C57BL/6 mice were infected intranasally with influenza virus A/PR/8/34. Pulmonary viral growth was determined at various days after infection by pfu experiment. Perforin-mediated apoptotic degeneration was observed by Immunohistochemical staining. LDH-release method was used for detection of specific CTL and NK cell activity from spleen cells.Results:Mice deficient in the perforin gene showed an increased virus growth and prolonged virus shedding. The appearance of apoptotic degeneration in virally infected lung cells was delayed in perforin-deficient mice. The cytolytic activities of natural killer cells and virus-specific cytotoxic T lymphocytes were significantly lower than that of wild-type mice.Conclusion:Perforin plays a critical role in the host defense system against primary influenza virus infection.

In Vitro Antitumor Activities and phenotype Analysis of Tumor—infiltrating Lymphocytes(TIL)and Lymphokine—activating Killer(LAK)Cells From Patients With Osteosarcoma / 中国免疫学杂志

TIL were isolated from the resected tumor mass,and lymphocytes were separated from pe-ripheral blood of 12 patients with osteosarcoma.In vitro antitumor activity and spesificity of rIL—2 activated TIL and LAK cells were determined by 4 hour ~(51)Cr releasing assay.Phenotypeanalysis of TIL were carried out in different intervals of incubation.The results revealed thatduring the incubation period from Day 15—Day 20,the difference between average cytolytic ac-tivities of TIL and LAK ceils with K562 and LiBr cells as target cells(E:T ratio 25:1)were in-significant,while that from 7 of those patients with autotumor cells as target cells were signifi-cant.The phenotype analysis showed that the percentage of CD3~+ cells remained constant,whilethat of CD4~+ cells tends to increase,and that of CD8~+cells tends to decrease during the whole in-cubation period.

In vitro antitumor mechanisms of human Lymphokine-Activated killer cells / 中国免疫学杂志

We previously demonstrated the cytotoxicity of human LAK cells to solid tumors in vitro. The work we reported here investigated the mechanisms involved in killing of solid tumor cells by LAK cells. It was shown that the cytotoxicity of LAK cells was mediated by some factors secreted by LAK cells and effects of direct contact with targets. Following lysis, the nucli of targets were destroyed and fragments of DNA were released into the medium. The cytotoxicity of LAK cells, under some circumtances, was of a positive correlation to the proliferation of themselves.