Histeroscopía quirúrgica: experiencia, resultados y complicaciones según clasificación Clavien-Dindo / Surgical hysteroscopy: experience, results and complications according to Clavien-Dindo classification

OBJETIVO: Describir y analizar la experiencia clínica, resultados y complicaciones según Clavien-Dindo de las histeroscopías quirúrgicas realizadas en pabellón. MÉTODOS: Estudio descriptivo retrospectivo de las histeroscopías quirúrgicas realizadas entre el 1 de enero de 2012 y 1 de enero de 2018 en el Hospital Clínico de la Universidad de Chile. RESULTADOS: Hubo 613 histeroscopías quirúrgicas en el período analizado, de las cuales 593 cumplieron con los requisitos para incluirse en este estudio. Las indicaciones para realizar el procedimiento fueron: pólipo endometrial (56,3%), miomas uterinos (22,1%), sangrado uterino anormal (4,3%) y otras (17,7%). Hubo un 89,2% de concordancia entre el diagnóstico intraoperatorio y el estudio histopatológico. Se pesquisaron 11 hiperplasias endometriales sin atipías, 3 con atipías y 10 neoplasias malignas. Cabe destacar que, del total de pólipos resecados, hubo 8 casos (2,5%) con potencial malignidad (atipías o neoplasia maligna). Según la clasificación Clavien Dindo, hubo 22 complicaciones intraoperatorias (3,7%) grado I o II, cuyo diagnóstico fue realizado en el acto quirúrgico. No hubo complicaciones grado III o más (severas, con reintervención). CONCLUSIÓN: La tasa de éxito, correlación histeroscópica - anatomopatológica final y complicaciones fue similar a lo publicado en la literatura disponible. El diagnóstico intraoperatorio de la lesión y su reparación en el mismo acto quirúrgico, disminuye el riesgo de morbimortalidad de las pacientes, haciéndolo similar al de una paciente sin complicación. Utilizar la clasificación Clavien Dindo para evaluar las complicaciones nos permitirá en adelante, objetivar, mejorar aspectos del procedimiento quirúrgico y plantear estrategias de prevención y manejo de dichos eventos adversos.

OBJECTIVE: To describe and analyze the clinical experience, results and complications according to Clavien-Dindo of surgical hysteroscopies performed in the ward. METHODS: Retrospective descriptive study of surgical hysteroscopies performed between January 1, 2012 and January 1, 2018 at the Hospital Clinico of the University of Chile. RESULTS: There were 613 surgical hysteroscopies in the analyzed period of which 593 fulfilled the requirements to be included in this study. The indications to perform the procedure were: endometrial polyp (56.3%), uterine fibroids (22.1%), abnormal uterine bleeding (4.3%) and others (17.7%). There was an 89.2% agreement between the intraoperative diagnosis and the histopathological study. Eleven endometrial hyperplasias without atypia, 3 with atypia and 10 malignant neoplasms were investigated. It should be noted that, of the total of resected polyps, there were 8 cases (2.5%) with potential malignancy (atypia or malignant neoplasm). According to the Clavien Dindo classification, there were 22 intraoperative complications (3.7%) grade I or II, the diagnosis of which was made during surgery. There were no grade III or more complications (severe, with reoperation). CONCLUSION: The success rate, final hysteroscopic-pathological correlation and complications was similar to that published in the available literature. The intraoperative diagnosis of the lesion and its repair in the same surgical act, reduces the risk of morbidity and mortality of the patients, making it similar to that of a patient without complication. Using the Clavien Dindo classification to assess complications will henceforth allow us to objectify, improve aspects of the surgical procedure and propose strategies for the prevention and management of such adverse events.

Limited mycobacterial infection of the liver as a consequence of its microanatomical structure causing restriction of mycobacterial growth to professional phagocytes.

Among sites of extrapulmonary growth of Mycobacterium tuberculosis, the liver is the least infected. Our data suggest that this is due to the complete restriction of mycobacterial growth to liver macrophages. Unlike in organs more persistently seeded by M. tuberculosis, in the liver the bacteria do not infect cell types other than professional phagocytes.

Donor cell persistence and activation-induced unresponsiveness of peripheral CD8+ T cells.

We studied the impact of the duration of donor cell persistence on CD8+ T cell responsiveness after adoptive transfer of antigen-expressing lymphoid cells. Naive or immunized female mice were treated by adoptive transfer of spleen cells from mice ubiquitously expressing a lymphocytic choriomeningitis virus-derived cytotoxic T lymphocyte (CTL) epitope (gp33-41) either alone or in combination with the male H-Y antigen providing additional antigenic CTL and T helper cell determinants. Low doses of male spleen cells (or sorted B cells) primed CTL, while high doses of the same cells rendered them unresponsive. CTL unresponsiveness induced by high numbers of male spleen cells was dependent upon prolonged persistence of antigen-expressing donor cells. Unresponsive CTL reverted to a state of activation when the duration of donor cell chimerism was limited. Memory CTL could be rendered unresponsive if antigen-expressing donor cells were allowed to persist. These results suggest that, irrespective of the type of antigen-presenting cell and the functional state of the responding T cell, activation and unresponsiveness can represent two different outcomes critically determined by quantitative and kinetic differences of antigen persistence.

Rapid neutrophil response controls fast-replicating intracellular bacteria but not slow-replicating Mycobacterium tuberculosis.

Being one of the first cells to invade the site of infection, neutrophils play an important role in the control of various bacterial and viral infections. In the present work, the contribution of neutrophils to the control of infection with different intracellular bacteria was investigated. Mice were treated with the neutrophil-depleting monoclonal antibody RB6-8C5, and the time course of infection in treated and untreated mice was compared by using intracellular bacterial species and strains varying in virulence and replication rate. The results indicate that neutrophils are crucial for the control of fast-replicating intracellular bacteria, whereas early neutrophil effector mechanisms are dispensable for the control of the slow-replicating Mycobacterium tuberculosis.

Autoimmune intestinal pathology induced by hsp60-specific CD8 T cells.

Due to their ubiquitous distribution and high degree of structural similarity, heat shock proteins (hsp) are potential target antigens in autoimmune diseases. Here, we describe induction of intestinal inflammation following transfer of hsp60-reactive CD8 T cells into mice. Inflammatory reactions were MHC class I dependent and developed primarily in the small intestine. IFN gamma and TNF alpha, as well as gut-derived hsp60, were elevated at sites of T cell infiltration. Intestinal lesions were drastically reduced in mice lacking receptors for TNF alpha. Pathology also developed in germ-free mice, indicating recognition of host-derived hsp60 by CD8 T cells. This report demonstrates that CD8 T cells with defined antigen specificity cause intestinal inflammation, emphasizing a link between infection and autoimmune disease.

Antigen persistence and time of T-cell tolerization determine the efficacy of tolerization protocols for prevention of skin graft rejection.

We studied antigen-specific T-cell tolerization therapy using skin transplantation across a defined minor histocompatibility antigen difference. Specific tolerization protocols using short-lived peptide or long-lived spleen cells presenting the peptide as antigen prevented graft rejection without immunosuppression when started before or as long as 10 days after transplantation. Peptide-induced T-cell tolerance was transient, and antigen presentation by the graft was not sufficient to maintain tolerance. In contrast, transfer of antigen-expressing lymphoid cells induced long-lasting tolerance correlating with donor cell chimerism. These findings show that antigen-specific tolerization can induce graft acceptance even when begun after transplantation and that long-term graft survival depends on persistence of the tolerizing antigen.

Viral and bacterial infections interfere with peripheral tolerance induction and activate CD8+ T cells to cause immunopathology.

We studied the impact of various infectious and proinflammatory agents on the induction of peripheral T cell tolerance. Adoptive transfer of CD8+ T cells from lymphocytic choriomeningitis virus (LCMV) T cell receptor transgenic mice into LCMV antigen transgenic mice expressing the LCMV glycoprotein epitope (gp) 33-41 under control of a major histocompatibility complex class I promoter led to efficient induction of peripheral tolerance after a period of transient activation. If, however, the recipient mice were challenged with viral or bacterial infections or proinflammatory agents (lipopolysaccharide or Poly:IC) early after cell transfer, tolerance induction was prevented and instead, CD8+ T cell activation leading to vigorous expansion and generation of cytolytic activity ensued. This became manifest in significant immunopathology mainly involving destruction of the splenic architecture and lysis of antigen-expressing lymphocyte and macrophage populations. Important parameters involved in the activation of host-reactive T cells by nonspecific infectious agents included the presence, localization, and quantity of the specific transgene-encoded self-antigen; in contrast, CD4+ T cells were not required. In mice surviving the acute phase, the transferred CD8+ T cells persisted at high levels in an anergic state; they were unable to generate cytolytic activity in vitro or to control LCMV infection in vivo. These results impinge on our understanding of the role of infectious agents in graft verus host reactions towards minor histocompatibility antigens.

Crucial role of marginal zone macrophages and marginal zone metallophils in the clearance of lymphocytic choriomeningitis virus infection.

Macrophages play a key role in the immune defense against pathogens. They control early invasion by antigen-unspecific phagocytosis of pathogens and act as professional antigen-presenting cells to induce antigen-specific T cell responses. To investigate the involvement of particular subsets of the splenic macrophages in an antiviral immune response, we selectively depleted mice of splenic marginal zone macrophages (MZM) and marginal zone metallophils (MM) using the clodronate liposome depletion technique. MZM- and MM-depleted mice were not able to control an infection with lymphocytic choriomeningitis virus (LCMV). In these mice, LCMV spread from the spleen to peripheral organs at an early phase of infection. The virus-specific cytotoxic T lymphocyte (CTL) response was induced initially, yet was exhausted in parallel with the overwhelming virus replication. These findings suggest that MZM and MM play a crucial role in the early control of a LCMV infection by preventing immediate virus spread to peripheral organs, but are not essential for the induction of the LCMV-specific CTL response.

CD5- CD8 alpha beta intestinal intraepithelial lymphocytes (IEL) are induced to express CD5 upon antigen-specific activation: CD5- and CD5+ CD8 alpha beta IEL do not represent separate T cell lineages.

We followed alpha beta T cell receptor (TCR) usage in subsets of gut intraepithelial lymphocytes (IEL) in major histocompatibility complex class I-restricted alpha beta TCR-transgenic (tg) mice. The proportion of tg alpha beta TCR+ CD8 alpha beta IEL is reduced compared with CD8+ splenocytes of the same animal, particularly under conventional conditions of maintenance. Further fractionation of CD8 alpha beta IEL according to the expression level of surface CD5 revealed that in conventionally housed animals tg TCR+ CD5- CD8 alpha beta IEL are as frequent as in specific pathogen-free (SPF) mice, whereas tg TCR+ CD5int or, even more pronounced, tg TCR+ CD5hi CD8 alpha beta IEL are greatly diminished when compared with mice kept under SPF conditions. Upon antigen-specific stimulation of CD5- CD8 alpha beta IEL in vitro, CD5 surface expression is up-regulated on a large fraction of cells within 48 h. Up-regulation of CD5 surface expression is further enhanced by the presence of the anti-alpha IEL monoclonal antibody 2E7. This clearly demonstrates that CD5-, and CD5+ CD8 alpha beta IEL cannot be considered as separate T cell lineages.

Peptide antigen treatment of naive and virus-immune mice: antigen-specific tolerance versus immunopathology.

Peptide-specific down-regulation of T cell responses may represent a powerful tool to intervene in autoimmune diseases or graft rejections. It is therefore important to know whether peptide treatment tolerizes both naive and antigen-experienced memory T lymphocytes. Here we show that a major histocompatibility complex class I binding peptide, derived from the glycoprotein (GP33 peptide) of lymphocytic choriomeningitis virus (LCMV), specifically tolerized naive cytotoxic T lymphocytes (CTL) when administered three times intraperitoneally in incomplete Freund's adjuvants. However, in the presence of GP33-specific memory CTL in LCMV-primed mice, the same treatment had a general immunosuppressive effect on unrelated third-party antigen-specific T cell responses and caused severe immunopathological damage to the spleen.

Antigen localisation regulates immune responses in a dose- and time-dependent fashion: a geographical view of immune reactivity.

This review summarises experimental evidence to illustrate that induction of immune reactivity depends upon antigen reaching and being available in lymphoid organs in a dose- and time-dependent manner. If antigen reaches lymph organs in a localised staggered manner and with a concentration gradient, a response is induced in the draining lymph node. Antigen-presenting cells are of critical importance to transport antigen from the periphery to local organised lymphoid tissue. If antigen is all over the lymphoid system, then it deletes all specific cells in the thymus or induces them within a few days; because of their limited life-span they then die off, leaving the repertoire depleted of this specificity. If antigen does not reach lymphoid organs it is ignored by immune cells. Once a response is induced, activated but not resting T cells will reach antigen outside lymphoid organs, whereas activated B cells differentiate into plasma cells in an inducing environment, mostly in lymphoid tissue including bone marrow, but also in chronic lymphoid-like infiltrations in peripheral organs. In immunopathology (when the infectious agent is known) or in autoimmunity (when the triggering infectious agent is not known or not recognised) lymphoid tissue may become organised close to the antigen (e.g. in organ-specific autoimmune diseases) and may thereby maintain an autoantigen-driven disease-causing immune response for a long time. The notion that native T cells get induced or silenced in the periphery may be questioned because induction can only occur in lymphoid organs providing anatomical structures where critical cell-cell interactions are properly guided and where, therefore, cells are likely to meet sufficiently frequently and in a critical milieu. Since overall immune reactivity critically depends upon the localisation of antigens in a dose- and time-dependent manner, it seems more likely-but this remains to be shown-that activated T cells may get exhausted in non-lymphoid peripheral tissues, whereas they are usually maintained in lymphoid organs. The critical role of antigen in regulating immune responses also has relevance for our understanding of immunological defence against epithelial and mesenchymal tumours, against many infectious diseases and for understanding autoimmunity and immunological memory. Collectively the data indicate that antigen, dependent upon localisation, dose and time, seems to be the simplest regulator of immune responses.

Bystander activation of cytotoxic T cells: studies on the mechanism and evaluation of in vivo significance in a transgenic mouse model.

Bystander activation, i.e., activation of T cells specific for an antigen X during an immune response against antigen Y may occur during viral infections. However, the low frequency of bystander-activated T cells has rendered it difficult to define the mechanisms and possible in vivo relevance of this nonspecific activation. This study uses transgenic mice expressing a major histocompatibility complex class I-restricted TCR specific for glycoprotein peptide 33-41 of lymphocytic choriomeningitis virus (LCMV) to overcome this limitation. CD8+ T cells from specific pathogen-free maintained, unimmunized "naive" TCR transgenic mice can differentiate into LCMV-specific cytolytic effector CTL during infections with vaccinia virus or Listeria monocytogenes in vivo or mixed lymphocyte culture in vitro. We show that in these model situations (a) nonspecifically activated CTL are able to confer antiviral protection in vivo, (b) bystander activation is largely independent of the expression of a second T cell receptor of different specificity, (c) bystander activation is not mediated by a broadly cross-reactive TCR, but rather by cytokines, (d) bystander activation can be mediated by cytokines such as IL-2, but not alpha/beta-IFN in vitro; (e) bystander activation is, overall, a rare event, occuring in vivo in roughly 1 in 200 of the LCMV-specific CTL during infection of TCR transgenic mice with vaccinia virus; (f) bystander activation does not have a significant functional impact on nontransgenic CTL memory under the conditions tested; and (g) even in the TCR transgenic situation, where unphysiologically high numbers of T cells of a single specificity are present, bystander activation is not sufficient to cause clinically manifest autoimmune disease in a transgenic mouse model of diabetes. We conclude that although bystander activation via cytokines may generate cytolytically active CTL from naive precursors, quantitative considerations suggest that this is usually not of major biological consequence.

A functional and kinetic comparison of antiviral effector and memory cytotoxic T lymphocyte populations in vivo and in vitro.

To analyze the critical parameters for effective antiviral cytotoxic T lymphocyte (CTL) activity in vivo, control of lymphocytic choriomeningitis virus (LCMV) infection in the spleen was studied after adoptive transfer of different spleen cell populations into preinfected recipients. The quantitative, qualitative and kinetic requirements for virus control were defined and related to in vitro assays to compare the antiviral protective function of CTL from naive, acutely infected and memory mice. Treatment of mice with an established but limited LCMV infection by adoptive transfer of spleen cells from acutely LCMV-infected mice led to complete virus elimination mainly mediated by donor-derived CD8+ T cell-mediated, perforin-dependent cytotoxicity. Since virus is continuously spreading and the number of infected target cells rapidly increases, the time until target cell lysis is achieved was critical: if release of viral progeny was not prevented early, additional time to perform effector function did not improve overall virus control. When the function of various cell populations was compared in this model, we found that CTL from naive and memory mice perform considerably less well than CTL from acutely infected mice. In vitro studies indicated that this is probably due to the fact that they can not fulfill the limiting time requirements for immediate antiviral protection: while CTL from acutely infected mice can perform lytic effector function immediately, memory CTL require a considerable reactivation time before they can lyse infected target cells. This reactivation does not necessarily involve cell division. These findings illustrate how critical time limitations are for CTL to mediate early control of a dynamic virus infection in vivo.

Immunity to viruses in B cell-deficient mice: influence of antibodies on virus persistence and on T cell memory.

Mice rendered B cell deficient by targeted disruption of the immunoglobulin mu chain gene (IgM-/- mice) were used to analyze the role of antibodies and B cells in viral infections; homozygous IgM-/- mice were bred in a way to avoid transmission of maternal antibodies. After infection with vesicular stomatitis virus (VSV), IgM-/- mice developed paralytic disease and subsequently died, whereas C57BL/6 control mice or IgM-/- mice passively protected with VSV-neutralizing antibodies survived. Furthermore, IgM-/- mice showed increased natural killer (NK) activity upon exposure to either lymphocytic choriomeningitis virus (LCMV) or to poly(I).poly(C), while NK activity in untreated IgM-/- mice was within normal ranges. Cytotoxic T cell responses were comparable in IgM-/- and control mice infected either with VSV or with vaccinia virus or with low doses of LCMV (10(2) infectious focus-forming units [ifu]). After intracerebral infection with LCMV-Armstrong, CD8+ T cell-mediated lethal lymphocytic choriomeningitis developed independently of the presence of B cells and antibodies. After infection with high doses (2 x 10(6) - 5 x 10(6) ifu) of LCMV-WE or LCMV-Docile, IgM-/- mice exhibited a reduced capacity to control these primary infections and had elevated virus titers for prolonged times (> 60 days). Nevertheless, the cytotoxic T cell response against LCMV in the early phase of infection was comparable in IgM-/- and control mice, but disappeared in those IgM-/- mice which had a persistent viral infection. Cytotoxic T cell memory was apparently unimpaired in low-dose-primed IgM-/- mice, which were able to control the primary virus infection; both IgM-/- and control mice cleared a high intravenous dose of virus within 2 days after challenge infection. This indicates that an efficient T cell memory against LCMV was established in the absence of B cells.

T cell priming versus T cell tolerance induced by synthetic peptides.

It is well known that synthetic peptides are able to both induce and tolerize T cells. We have examined the parameters leading either to priming or tolerance of CD8+ cytotoxic T lymphocytes (CTL) in vivo with a major histocompatibility complex class I (H-2 Db) binding peptide derived from the glycoprotein (GP aa33-41) of lymphocytic choriomeningitis virus (LCMV). By varying dose, route, and frequency of LCMV GP peptide application, we found that a single local subcutaneous injection of 50-500 micrograms peptide emulsified in incomplete Freund's adjuvant protected mice against LCMV infection, whereas repetitive and systemic intraperitoneal application of the same dose caused tolerance of LCMV-specific CTL. The peptide-induced tolerance was transient in euthymic mice but permanent in thymectomized mice. These findings are relevant for a selective use of peptides as a therapeutic approach: peptide-induced priming of T cells for vaccination and peptide-mediated T cell tolerance for intervention in immunopathologies and autoimmune diseases.

Escape of thymocytes and mature T cells from clonal deletion due to limiting tolerogen expression levels.

Expression of self antigen on lymphohemopoietic cells and in the thymus has been shown to cause tolerance by negative selection. To investigate the role of self antigen expression levels on the induction of tolerance, we generated transgenic mice expressing the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) driven by the H-2Kb promoter. Two mouse lines differing in transgene expression levels were obtained and evaluated for the induction of tolerance to LCMV-GP. LCMV-GP high (GPhi) expressing animals thymically deleted self-reactive thymocytes. Low expressors (GPlo) partially deleted self-reactive mature T cells in the periphery in the absence of any obvious signs of negative selection in the thymus. Functionally, the LCMV-GP-specific cytotoxic T cell (CTL) response was absent in GPhi mice, whereas GPlo mice produced diminished LCMV-GP-specific CTL responses. Therefore limiting levels of expression of self antigen influence efficiency of negative selection, enabling potentially self-reactive T cells to escape from tolerance induction.

Peptide-induced T-cell tolerance to prevent autoimmune diabetes in a transgenic mouse model.

A synthetic peptide corresponding to an immunodominant epitope of lymphocytic choriomeningitis virus glycoprotein (LCMV GP) was used to prime or to tolerize CD8+ T cells in vivo, dependent on mode of immunization. Peptide-specific tolerance was then examined in transgenic mice expressing LCMV GP in the beta islet cells of the pancreas; these mice develop CD8+ T-cell-mediated diabetes within 8-14 days after LCMV infection. Specific peptide-induced tolerance prevented autoimmune destruction of beta islet cells and diabetes in this transgenic mouse model.

T cell immunity after a viral infection versus T cell tolerance induced by soluble viral peptides.

The fate of in vivo activated CD8+ cytotoxic T cells was studied in transgenic mice expressing a T cell receptor (TCR) specific for the lymphocytic choriomeningitis virus (LCMV) glycoprotein peptide 33-41 presented by major histocompatibility complex (MHC) class I molecules. LCMV infection of TCR transgenic mice induced LCMV-specific effector and memory T cells whereas injection of soluble LCMV glycoprotein peptide 33-41 resulted in tolerance by peripheral deletion and anergy of LCMV-specific T cells after an initial expansion phase. Similarly, LCMV peptide 33-41-specific tolerance could be achieved in normal C57BL/6 mice and was not abrogated by an LCMV infection. These results obtained with a classically MHC-restricted peptide antigen parallel previous findings with retroviral or bacterial superantigens and indicate a possibility to modulate specifically mature peripheral cytotoxic T lymphocytes in vivo.

Induction of diabetes is influenced by the infectious virus and local expression of MHC class I and tumor necrosis factor-alpha.

To study self reactivity, a transgenic mouse model has been established in which the lymphocytic choriomeningitis virus (LCMV) glycoprotein (gp) is expressed in the beta-islet cells of the pancreas (rat insulin promoter (RIP)-gp). These mice (H-2b) do not spontaneously develop diabetes; however, infection with the LCMV strain WE rapidly induces hyperglycemia. In this study, comparative analysis of H-2k RIP-gp-transgenic animals demonstrated that the haplotype influences the incidence and kinetics of diabetes and alters the requirement for the CD4+ T cell subset. This study also showed that the properties of the virus expressing the self target Ag determined whether hyperglycemia occurred in RIP-gp-transgenic mice. Various LCMV strains were able to induce diabetes in RIP-gp-transgenic animals, whereas infection with a recombinant vaccinia virus expressing LCMV-gp (vacc-gp) did not induce diabetes. However, vacc-gp could induce diabetes in double (RIP-gp/TCR)-transgenic mice, where the majority of CD8+ T cells expressed a receptor specific for LCMV-gp, suggesting that a critical number of self-reactive T cells must be activated to induce disease. Notably, histologic analysis of pancreata taken various days after LCMV or vacc-gp infections indicated that induction of diabetes coincided with an increase in MHC class I expression on the islets of Langerhans. Additional studies with vacc-gp were done to determine other factors that possibly enhance autoimmune attack. Transgenic mice expressing both LCMV-gp and TNF-alpha under the control of the RIP were infected with vacc-gp, and 50% of RIP-gp/TNF-alpha-transgenic animals became hyperglycemic. These data suggest that the increased local lymphocyte traffic as a result of TNF-alpha expression attracts activated gp-specific T cells, enhancing the possibility of hyperglycemia. Collectively, these results demonstrate that the induction of diabetes in this model is influenced by the MHC haplotype, the infectious agent, TNF-alpha expression, the level of MHC class I expression, and the induction of a threshold number of self-reactive CTL.