Laparoscopic sacropexy: a retrospective analysis of the subjective outcome in 310 cases.

Background and Objective. The aim of this study was to assess the subjective outcome following laparoscopic sacropexy. Methods. We performed a retrospective cohort study among women treated for descensus with laparoscopic sacropexy between January 2000 and December 2007. 310 patients received questionnaires during followup assessing major pre- and postoperative symptoms and overall satisfaction. Results. 214 (69%) patients responded to the questionnaire. Mean followup was 24.5 months. The number of patients with back or lower abdominal pain, foreign body sensation in the vagina and prolapse-related symptoms, urinary symptoms, vaginal and bladder infections, and the need for pessary usage decreased significantly postsurgically. Bowel symptoms increased slightly but not significantly. Two years after surgery, nearly 2 thirds of the women were satisfied or very satisfied with the outcome. Conclusion. Laparoscopic sacropexy is an effective treatment of descensus, with favorable or satisfactory subjective outcomes.

Divergent therapeutic and immunologic effects of oligodeoxynucleotides with distinct CpG motifs.

Immune stimulatory oligodeoxynucleotides (ODN) with unmethylated CpG motifs are potent inducers of both innate and adaptive immunity. It initially appeared that a single type of optimal CpG motif would work in all applications. We now report that specific motifs of CpG ODN can vary dramatically in their ability to induce individual immune effects and that these differences impact on their antitumor activity in different tumor models. In particular, a distinct type of CpG motif, which has a chimeric backbone in combination with poly(G) tails, is a potent inducer of NK lytic activity but has little effect on cytokine secretion or B cell proliferation. One such NK-optimized CpG ODN (1585) can induce regression of established melanomas in mice. Surprisingly, no such therapeutic effects were seen with CpG ODN optimized for activation of B cells and Th1-like cytokine expression (ODN 1826). The therapeutic effects of CpG 1585 in melanoma required the presence of NK but not T or B cells and were not associated with the induction of a tumor-specific memory response. In contrast, CpG 1826, but not CpG 1585, was effective at inducing regression of the EL4 murine lymphoma; this rejection was associated with the induction of a memory response and although NK cells were necessary, they were not sufficient. These results demonstrate that selection of optimal CpG ODN for cancer immunotherapy depends upon a careful analysis of the cellular specificities of various CpG motifs and an understanding of the cellular mechanisms responsible for the antitumor activity in a particular tumor.

Lactoferrin binds CpG-containing oligonucleotides and inhibits their immunostimulatory effects on human B cells.

Unmethylated CpG dinucleotide motifs in bacterial DNA, as well as oligodeoxynucleotides (ODN) containing these motifs, are potent stimuli for many host immunological responses. These CpG motifs may enhance host responses to bacterial infection and are being examined as immune activators for therapeutic applications in cancer, allergy/asthma, and infectious diseases. However, little attention has been given to processes that down-modulate this response. The iron-binding protein lactoferrin is present at mucosal surfaces and at sites of infection. Since lactoferrin is known to bind DNA, we tested the hypothesis that lactoferrin will bind CpG-containing ODN and modulate their biological activity. Physiological concentrations of lactoferrin (regardless of iron content) rapidly bound CpG ODN. The related iron-binding protein transferrin lacked this capacity. ODN binding by lactoferrin did not require the presence of CpG motifs and was calcium independent. The process was inhibited by high salt, and the highly cationic N-terminal sequence of lactoferrin (lactoferricin B) was equivalent to lactoferrin in its ODN-binding ability, suggesting that ODN binding by lactoferrin occurs via charge-charge interaction. Heparin and bacterial LPS, known to bind to the lactoferricin component of lactoferrin, also inhibited ODN binding. Lactoferrin and lactoferricin B, but not transferrin, inhibited CpG ODN stimulation of CD86 expression in the human Ramos B cell line and decreased cellular uptake of ODN, a process required for CpG bioactivity. Lactoferrin binding of CpG-containing ODN may serve to modulate and terminate host response to these potent immunostimulatory molecules at mucosal surfaces and sites of bacterial infection.

Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo.

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN synthesized with a nuclease-resistant phosphorothioate backbone have been shown to be potent Th1-directed adjuvants in mice, but these motifs have been relatively inactive on primate leukocytes in vitro. Moreover, in vitro assays that predict in vivo adjuvant activity for primates have not been reported. In the present study we tested a panel of CpG ODN for their in vitro and in vivo immune effects in mice and identified in vitro activation of B and NK cells as excellent predictors of in vivo adjuvant activity. Therefore, we tested >250 phosphorothioate ODN for their capacity to stimulate proliferation and CD86 expression of human B cells and to induce lytic activity and CD69 expression of human NK cells. These studies revealed that the sequence, number, and spacing of individual CpG motifs contribute to the immunostimulatory activity of a CpG phosphorothioate ODN. An ODN with a TpC dinucleotide at the 5' end followed by three 6 mer CpG motifs (5'-GTCGTT-3') separated by TpT dinucleotides consistently showed the highest activity for human, chimpanzee, and rhesus monkey leukocytes. Chimpanzees or monkeys vaccinated once against hepatitis B with this CpG ODN adjuvant developed 15 times higher anti-hepatitis B Ab titers than those receiving vaccine alone. In conclusion, we report an optimal human CpG motif for phosphorothioate ODN that is a candidate human vaccine adjuvant.

CpG motifs in bacterial DNA activate leukocytes through the pH-dependent generation of reactive oxygen species.

B cells and monocytes endocytose DNA into an acidified intracellular compartment. If this DNA contains unmethylated CpG dinucleotides in particular base contexts (CpG motifs), these leukocytes are rapidly activated. We now show that both B cell and monocyte-like cell line responses to DNA containing CpG motifs (CpG DNA) are sensitive to endosomal acidification inhibitors; they are completely blocked by bafilomycin A, chloroquine, and monensin. The specificity of these inhibitors is demonstrated by their failure to prevent responses to LPS, PMA, or ligation of CD40 or IgM. Acidification of endosomal CpG DNA is coupled to the rapid generation of intracellular reactive oxygen species. The CpG DNA-induced reactive oxygen species burst is linked to the degradation of IkappaB and the activation of NFkappaB, which induces leukocyte gene transcription and cytokine secretion. These studies demonstrate a novel pathway of leukocyte activation triggered by CpG motifs.

Differential activation of Th1 and Th2 CD4+ cells by murine brain microvessel endothelial cells and smooth muscle/pericytes.

CD4+ Th cell infiltration into the brain and the activation by cellular elements of the central nervous system (CNS) are thought to be important steps in the initiation of CNS autoimmune diseases. T cell activation requires Ag-specific stimulation and additional costimulatory signals provided by the APC. Here we describe how murine brain microvessel endothelial (En) cells and smooth muscle/pericytes (SM/P) selectively induce the Ag-specific activation of different Th1 and Th2 CD4+ T cell clones. Th1 and Th2 cell clones were used that were specific for the same peptide Ag in the context of the same class II allotype. SM/P preferentially activated Th1 cell clones, whereas En cells activated Th2 cell clones better, as reflected by cell proliferation and production of IL-2 by SM/P-activated Th1 clones and IL-4 by Th2 clones. There was no difference in the level of expression of CD4, CD2, or LFA-1 molecules between these Th cell clones, and anti-CD4, CD2, LFA-1 or ICAM-1 mAb did not differentially affect Ag-induced proliferation among the clones. Moreover, antibody to CD28 did not influence Ag presentation by brain microvessel En or SM/P cells to Ag-specific Th1 and Th2 clones. These results suggest that: 1) different The subsets might require different signals for their activation; 2) different APC might provide different costimulatory signals for Th cell subsets; and 3) brain microvessel En and SM/P might play a differential role in induction of autoreactive T cell responses in the CNS.

Adhesion molecules on murine brain microvascular endothelial cells: expression and regulation of ICAM-1 and Lgp 55.

The mechanisms for the initiation of immune reactions in the central nervous system are poorly understood. In this report, we describe the presence of intercellular adhesion molecule-1 (ICAM-1) and Lgp 55 (suggested mouse homologue of human intercellular adhesion molecule-2, ICAM-2) on the surface of brain microvessel endothelium (EN) cells and show in vitro induction of ICAM-1 molecules on EN cells with pro-inflammatory cytokines. ICAM-1 expression was detected using flow cytometry analysis with biotinylated anti-ICAM-1 antibody (YN1/1.7.4). Lgp 55 expression was characterized using PA3 monoclonal antibody. According to our results, 30-40% of the non-activated brain EN cells expressed ICAM-1 and 15-20% expressed Lgp 55 molecules. The ICAM-1 molecule expression was increased after the activation of the cells with recombinant murine gamma interferon (IFN-gamma), tumor necrosis factor (TNF-alpha), and interleukin-1 alpha (IL1-alpha) in a dose-dependent manner. The increased ICAM-1 expression was detected as early as 2 h following the cytokine treatment and reached its maximum after 24 h. Transforming growth factor-beta (TGF-beta) did not influence the expression of ICAM-1 molecule. Lgp 55 molecule does not seem to be regulated by pro-inflammatory cytokines. ICAM-1 and Lgp 55 expression was found to be polarized on the luminal surface of EN by confocal laser microscopy suggesting accessibility for leukocytes. Inducible ICAM-1 expression may play a critical role in formation of inflammatory reactions inside the central nervous system.

Adhesion of splenocytes to brain microvascular endothelium in the BALB/c and SJL/j mouse systems.

Adhesion of hematopoietic cells to endothelial (En) cells plays an important role in their migration into extravascular tissue. This report characterizes the adhesion properties of naive splenocytes to syngeneic and allogeneic mouse brain microvascular endothelium isolated from the BALB/c or SJL/j mouse strains. Syngeneic adhesion reaches maximum levels by 60 min at 37 degrees C, but is more pronounced in the BALB/c system (mean adhesion = 10.7% +/- 1.0) compared to adhesion seen in the SJL/j (mean adhesion = 4.3% +/- 0.6). BALB/c, but not SJL/j adhesion, seems to be mediated, at least in part, by the interaction of CD11a/CD18 (lymphocyte function-associated antigen 1 (LFA-1] with one of its ligands, because BALB/c adhesion is partially inhibited when the assay is carried out either in the presence of chelating agents or with antibodies to the CD11a/CD18 molecule. Activation of the endothelium with recombinant interferon-gamma (rIFN-gamma), recombinant interleukin-1 alpha (rIL-1 alpha), and recombinant tumor necrosis factor-alpha (rTNF-alpha), enhances adhesion in both BALB/c and SJL/j. IFN-gamma and IL-1 alpha mediated adhesion enhancement is abrogated by antibodies to the CD11a/CD18 molecules in the BALB/c but not in the SJL/j system. The adhesion of splenocytes to mouse brain En clearly has unique properties, and whether or not the differences seen in the SJL/j system in any way influences its susceptibility to the autoimmune demyelinating disease, experimental autoimmune encephalitis, remains to be determined.

Activation of CD4+ lymphocytes by syngeneic brain microvascular smooth muscle cells.

Splenocyte proliferation as measured by [3H]thymidine incorporation was detected when brain microvessel smooth muscle cells (SM) were cocultured with syngeneic spleen cells. This report focuses on the role of different lymphocyte populations in this activation. The central role of CD4+ T cells in the proliferation response has been established by different sets of experiments. The phenotypic characterization of splenic lymphocytes before and after the co-culture showed that the only cell type present in higher number after the co-culture than before is the CD4+ T cell. When CD4+ cells were purified by flow microfluorimetry and co-cultured with SM a strong proliferative response was detected. In contrast, purified CD8+ cells in co-culture with SM cells did not proliferate. The activation of CD4+ cells by SM required direct cell-to-cell contact and could be detected on the fourth day, reaching maximal levels at the 6th and 7th days of the co-culture. The activation is more pronounced in the syngeneic system than under allogeneic conditions and is inhibited by anti-MHC II mAb, but not by anti-MHC II mAb. The finding that vascular smooth muscle cells can activate syngeneic T cells may have important implications concerning the mechanism of induction of vasculitis.

Antigen presentation by brain microvessel smooth muscle and endothelium.

It has been previously reported that cultured brain microvessel smooth muscle cells (SM) express major histocompatibility complex (MHC) class II antigen. Here we report that SM is able to present ovalbumin (OVA) antigen to an OVA-specific T cell hybridoma (A2.2E10) and also presents keyhole limpet hemocyanin (KLH) to a KLH-specific T cell clone (HDK-1). Both the class II expression and the antigen-presenting capacity of SM cells is increased by interferon-gamma stimulation. Antigen presentation by SM is also MHC restricted as it is blocked by anti-Ia monoclonal antibodies. In contrast to SM, brain endothelium (En) presents whole OVA, digested OVA and KLH poorly, to a much lesser degree than SM, to the same antigen-specific T cells.

Experimental autoimmune inflammatory myopathy.

We report an experimental model of autoimmune inflammatory myopathy. Splenic cells from two inbred murine strains (BALB/c and SJL/J) are activated (immunized) in vitro by co-culture with their respective syngeneic skeletal muscle myotubes. Subsequent injection of the activated splenocytes with or without B. pertussis into the respective syngeneic hosts results in inflammatory myopathy in the SJL/J mice but never in the BALB/c mice. The muscle inflammation is very similar in appearance to human autoimmune inflammatory myopathies. The myositis is not effector cell-skeletal muscle specific because splenocytes activated by co-culture with smooth muscle will also elicit skeletal muscle lesions. Both strains of skeletal muscle appear to express class II (Ia) antigens and the splenocytes from both strains appear to be equally activated. Thus we postulate that the difference in the expression of myositis between the two strains is in the effector phase of the disease. Since SJL/J mice have vasoactive amine sensitive vascular systems and BALB/c do not, it is likely that activated splenocytes emigrate from muscle microvessels in the SJL/J strain whereas they cannot do so in the BALB/c strain. The most significant contribution of this model may be in its potential for addressing a sine qua non of cellular autoimmune disease, i.e. lymphocyte migration from the vascular compartment into the target tissue. Finally, the data support a cellular more than a humoral pathogenesis in this model.

Brain microvascular smooth muscle expresses class II antigens.

Mouse (BALB/c) splenic lymphocytes co-cultured in vitro with syngeneic brain-derived microvascular smooth muscle (SM) proliferate and become activated. After subsequent transfer of the activated lymphocytes to a syngeneic host, a vasculitis develops in the host. Investigation of the possible antigen-presenting properties of the cultured SM has resulted in the demonstration of class II (Ia) antigens on the SM. Fluorescence-activated cell sorter analysis has shown that an average of 31% of unstimulated SM cells in culture were positive when stained with an anti-IE of the appropriate haplotype (H2d), and an average of 20% were positive with an anti-IA of the H2d haplotype. Controls consisting of irrelevant antibodies of the same isotype, as well as an anti-IA of the H2s haplotype, were negative. In contrast, BALB/c-derived brain microvascular endothelial cells showed considerably less class II antigen expression (7% for both IA and IE).

Injuries of the diaphragm.

Injuries of the diaphragm in 86 patients occurring over a 10-year period were retrospectively reviewed. Blunt trauma victims experienced injury on the right and left with nearly equal frequency, representing a strikingly different experience from those reporting before 1970 when left-sided injuries predominated. Patients' complaints and physical findings were not reliable indicators of diaphram injury, but were usually manifestations of associated injury. Ninety-five per cent of our acute victims had other injuries. Routine chest X-rays were the most reliable diagnostic tools, yet these were normal in 1/4 of the patients. Diagnosis depends on high index of suspicion before operation and careful inspection of the diaphragm at operation. Initial thoracotomy required subsequent laparotomy to complete management in seven of 15, whereas laparotomy required supplemental thoracotomy only once in 65 instances. The superior operative approach, therefore, for either right or left diaphragmatic injury is initial laparotomy.