Contraception: Endometrial microvascular density during the normal menstrual cycle and following exposure to long-term levonorgestrel

The mechanisms that underlie progestogen-induced endometrialbreakthrough bleeding are poorly understood. The aim of thepresent study was to quantify endometrial microvascular densityin 54 controls and 42 women with 3–12 months' exposureto Norplant (levonorgestrel subdermal contraceptive implant)and to correlate it with bleeding pattern, endometrial histology,and peripheral plasma oestradiol and progesterone concentrations.Endometrial biopsies were processed routinely and sections immunostainedusing anti-CD34 antibody to identify vascular endothelial cells.Menstrual record card data were analysed using World HealthOrganization definitions. The mean microvascular density (±SEM) for control samples was 186 ± 8 vessels/mm², andthere were no significant differences across the cycle. Norplantuser's endometrial microvascular density was significantly elevatedabove controls (294 ± 18 vessels/mm², P = 3.36 x 10^–8).Endometrial microvascular density in Norplant users did notcorrelate with oestrogen concentrations prior to biopsy, bleedingpatterns or endometrial histology. The results from this studyshow that women receiving Norplant have significantly increasedendometrial microvascular density compared to controls. Anotherfinding from this study was that bleeding in Norplant usersoften occurred from thin atrophic endometrium. These resultsprovide new insights into the physiological mechanisms thatmay be involved in progestogen-induced endometrial bleeding.     

Uterus and endometrium: Microvascular density in conditions of endometrial atrophy

Irregular menstrual bleeding in users of hormonal contraceptionrepresents the single major reason for discontinuation of thesecontraceptive methods. The mechanisms which underlie these bleedingdisturbances are poorly understood, but appear to be associatedwith changes in the endometrial microvasculature following abnormalpatterns of sex steroid exposure. Endometrial microvasculardensity is known to be increased in users of the low-dose levonorgestrelcontraceptive implant, Norplant^®. This study explores microvasculardensity in other conditions of spontaneous (post-menopausal)and induced (danazol and goserelin) endometrial atrophy. Endometrialbiopsies were fixed, paraffin-embedded and sections were immunostainedusing anti-CD34 antibody to identify vascular endothelial cells.The mean microvascular density (±SEM) for control sampleswas 186±8 vessels/mm². There were no statistically significantchanges in vascular density observed across the menstrual cyde.Mean microvascular density in spontaneous and induced endo metrialatrophy did not differ significantly from that observed in thecontrol population. The mean micro- vascular density was 230±17vessels/mm² in 31 post-menopausal women, 269±67 vessels/mm²in25 subjects treated with danazol was and 191±45 vessels/mm²in nine subjects treated with goserelin. These findings suggestthat the mechanisms controlling microvascular density in conditionsof endometrial atrophy may vary according to the nature of theatrophic stimulus.     

Uterus and endometrium: Effect of high dose progestogens on white cells and necrosis in human endometrium

Endometrium was collected from 20 high-dose progestogentreatedpatients and examined for leukocyte populations by immunohistochemistryand phloxlne-tartrazine staining. A labelled streptavidin-biotin-alkalinephosphatase technique was used with antibodies against leukocytecommon antigen (LCA), T cells (CD₃) neutrophils (NP₅₇) macrophages (CD₃ KP1) and B cells (CD₃) The numbers of LCA (1070±117/mm²CD₃ (459±60/mm²), CD₆₈ (129±21/mm² positive cellsand endometrial granulated lymphocytes (EGL) (236±41/mm²were significantly higher than those in the control group (P<0.001). Of these, EGL increased most (6.7 times). NP₅₇ positive(NP₅₇₊) neutrophils were present in five out of 20 progestogentreated samples and NP₅₇₊ negative (NP_57– neutrophilsin another six out of 20; while a neutrophil was only identifiedin one control tissue (P=0.002). Three progestogen exposed endometrialsamples had either focal or extensive necrosis, and many NP₅₇₊and NP_57– neutrophils were present in the necrotic areas.EGL, neutrophils and macro phages are known to release a numberof cytolytic and cell toxic molecules which may play a rolein the initiation or acceleration of progestational endometrialnecrosis.     

The diagnosis of male infertility--prospective time-specific study of conception rates related to seminal analysis and post-coital sperm--mucus penetration and survival in otherwise unexplained infertility

Infertile women without any inherent female infertility factorsand able to secrete normal cervical mucus were studied prospectivelyin relation to post-coital sperm—mucus penetration (PCT)and their partner's seminal analysis, excluding men with azoospermia.Time-specific cumulative conception rates calculated as forlife-table analysis were related to each measured seminal variableon routine analysis of 2–3 samples (volume, density, proportionwith progressive motility, and proportion with normal morphology);to various derivatives from combinations of these variables;to seminal findings after vital staining; and to the PCT results.The best seminal predictor of fertility was the motile normalsperm density (MNSD), the 18 month conception rates being 57.4%+ 4.6 (SE) and 30.2% + 5.9 (ratio 1.9, P < 0.001) above andbelow a derived threshold value of 4 x 10⁶/ml. The PCT led torates of 55.6% ± 4.3 and 14.9% ± 5.1 (ratio 3.73,P < 0.001) for positive and negative results, respectively.The PCT also gave rise to a significantly distinct intermediatepoor-psitive sub-group (conception rate 30.6% ± 9.0).Seminal analysis (the MNSD) did not affect the conception rateassociated with a positive PCT but helped to discriminate furtherwith a negative PCT (conception rates 22.5% ± 8.7 withan MNSD above 4 x 10⁶/ml versus 5.6% ± 4.8 below, P <0.05). The PCT was the single best predictor of fertility butseminal analysis (the MNSD) was of additional value after anegative PCT.     

T cell subpopulation phenotypes in filarial infections: CD27 negativity defines a population greatly enriched for Th2 cells

Three-color flow cytometric analysis was used to define surfacemarkers which identify the T_h2-type CD4⁺ cells responsible forthe eosinophilia and elevated serum IgE typical of tissue invasivehelminth infections. A group of six mAba to well known cellsurface markers were screened for differential expression onCD4⁺ CD45RO⁺ lymphocytes from normal individuals (NL; n = 6)and filaria-infected patients (PT; n = 10). The majority ofmarkers were expressed equally by both groups, but the CD4⁺CD45RO⁺ cells in the PTs showed significantly higher levelsof expression of HLA-DR than those of NLs (P = 0.014). ThisCD4⁺ HLA-DR⁺ subpopulation was then studied further for itsexpression of an additional 10 activation and adhesion molecules.CD27 showed a trend for lower intensities of expression on PTCD4⁺ HLA-DR⁺ cells than on those of NLs. Analysis of the serumfrom both NLs and PTs revealed that PTs had significantly higherlevels of soluble CD27 and CD25 (IL-2R) In the serum than NLs(P < 0.01 and P = 0.022 respectively) indicating a generalstate of Immune activation and differentiation. Functional analysisof the CD4⁺ HLA-DR⁺ and the CD4⁺ CD27^– subpopulatlonsrevealed that the CD4⁺ HLA-DR⁺ cells produced significantlyhigher levels of IL-5 than the CD4⁺ HLA-DR^– cells (P <0.04), and the CD4⁺ CD27^– cells produced significantlyhigher levels of both IL-4 and IL-5 than the CD4⁺ CD27^–cells (P <0.05 and P <0.001 respectively). Thus, whilethe CD4⁺ CD27^– and CD27⁺ subpopulatlons contain T_h1 andT_h0 cells, only the CD4⁺ CD27^– population contains theT_h2 cells (producing both IL-4 and IL-5).     

An aggressive philosophy in controlled ovarian stimulation cycles increases pregnancy rates

To assess the effect of timing of human chorionic gonadotrophin(HCG) administration in ovarian stimulation cycles, the serumoestradiol concentration and follicle profile were comparedwith the clinical pregnancy rate in 582 ovarian stimulation— intra-uterine insemination (OS—IUI) cycles and3917 in-vitro fertilization—embryo transfer (IVF—ET)cycles. The pregnancy rates increased exponentially with increasingoestradiol in both OS—IUI and IVF—ET cycles (R²= 0.720, P < 0.001) but then decreased in OS-IUI cycles whenthe oestradiol concentration exceeded 5000 pmol/l (R² = 0.936,P < 0.004) at HCG administration. In OS—IUI cyclesthe percentage of cycles with three or more mature follicles( 18 mm diameter) increased up to an oestradiol concentrationof 5000 pmol/l then declined, mirroring the pregnancy rate (R²= 0.900, P = 0.01). The exponential increase in pregnancy ratewith increasing oestradiol concentration in IVF—ET cyclessuggests that high oestradiol concentration does not have adeleterious effect on endometrial receptivity. The decreasein pregnancy rate in OS-IUI cycles when oestradiol concentrationexceeded 5000 pmol/l reflected fewer mature follicles, resultingfrom premature administration of HCG to avoid severe ovarianhyperstimulation syndrome (OHSS). We recommend that HCG administrationbe delayed until multiple follicles have reached maturity, andreducing the risk of severe OHSS by converting high risk OS—IUIcycles to IVF—ET, or if funds or facilities are unavailable,transvaginally draining all but four or five mature follicles.     

Selection of intestinal intraepithelial lymphocyte T cell receptors: evidence for a dynamic tissue-specific process

An extensive comparison of TCRß V-reglon usage byCD8ß^-CD4⁺CD8⁺ intraepithelial lymphocytes (IEL), CD4^-CD8⁺IEL, and lymph node (LN) T cell subsets in three minor lymphocytestimulating (MIs)-disparate, MHC-ldentical mouse strains revealednovel TCR selection patterns. In cases where forbidden V regionswere expressed by CD8ß^- CD4^-CD8⁺ IEL, the same TCRswere deleted from CD8ß^– CD4⁺CD8⁺ IEL, Indicatingthat lack of CD8ß expression was not solely responsiblefor forbidden V-region expression. These results also suggestedthat CD4 may be involved in negative selection of CD4⁺CD8⁺ IELTCRs. In C57BR/cdJ (Mls-1^b2^b) mice, a major increase in V_ß3⁺CD4⁺CD8⁺IEL but not in other IEL or LN subsets was noted suggestinga subset-specific expansion of V_ß3⁺ cells. Negativeselection of V_ß14⁺ cells in only the CD4⁺CD8⁺ IELsubset further supported the existence of intestine-specificTCR selection processes. Analysis of V-reglon expression ofCD8ß⁺ and CD8ß^–CD4^–CD8⁺ IELsubsets revealed that forbidden V-region expression was notstrictly confined to the CD8ß^– subset in allcases. Overall, the data point to a dynamic, gut-specific TCRselection process that may be antigen driven.     

Endocrinology: Efficacy of chronic therapy with the gonadotrophin releasing hormone agonist decapeptyl in patients with polycystic ovary syndrome

Our objective was to assess the endocrine and morphologicalresponse of polycystic ovary syndrome (PCOS) in patients receiving6 months of therapy with the long-acting gonadotrophin releasinghormone agonist (GnRH agonist) decapeptyl (3.75 mg monthly injections).Eighteen documented PCOS patients were basally evaluated forhirsutism, gonadotrophin and androgen concentrations and ovarianmorphology using trans-vaginal ultrasonography. Measurementswere repeated at 3 and 6 months. The results (values as x ±SD) showed a significant improvement in hirsutism (Ferrimanscore 11.0 ± 5.9 versus 6.6 ± 2.7, P < 0.01),acne and seborrhoea. A significant post-treatment decrease ingonadotrophins [follicle-stimulating hormone (FSH): 5.8 ±1.8 versus 3.8 ± 1.1 IU/I, P < 0.01; luteinizing hormone(LH): 10.8 ± 8.3 versus 3.4 ± 3.3 IU/1, P <0.01], LH/FSH ratio (1.8 ± 1.1 versus 0.8 ± 0.6,P < 0.01) and androgen concentrations (free testosterone:4.0 ± 1.9 versus 1.9 ± 0.7 pg/ml, P < 0.01,₄-androstenedione: 3.9 ± 1.2 versus 1.9 ± 0.6ng/ml, P < 0.001) was also found, while oestradiol approximatedcastration concentrations (68.4 ± 29.5 versus 29.1 ±6.7 pg/ml, P < 0.001). Finally, mean ovarian volume (19.7± 6.2 versus 10.9 ± 4.6 cm³, P < 0.001), capsulethickness (2.5 ± 0.8 versus 1.9 ± 0.7 mm, P <0.05) and stromal density dropped significantly, as did uterinevolume (34.2 ± 10.5 versus 19.9 ± 8.9 cm3, P <0.01). In conclusion, treatment of our PCOS patients for 6 monthswith the GnRH agonist decapeptyl proved efficient in inducingsignificant clinical, biochemical and ovarian morphologicalimprovement.     

Epidermal growth factor receptors in human corpora lutea during the menstrual cycle and pregnancy

We have demonstrated the presence of epidermal growth factor(EGF) and its receptors in human non-gestational corpora lutea.To determine further the characteristics of EGF receptor binding,we examined 30 human corpora lutea throughout the luteal phaseand during pregnancy. Scatchard plots of EGF binding in 29 ofthe 30 corpora lutea were curvilinear, suggesting negative co-operativity.The mean ± SE of the association constant K_a was (0.9± 0.2) x 10⁹ 1/mol, the dissociation constant K_d was(2.2 ± 0.3) x10^–9 mol/1 and the number of bindingsites (Rt) was (15.8 ± 2.1) x10^–19 mol/µgprotein for non-gestational corpora lutea. The K_d increasedsignificantly in late pregnancies compared to early pregnancies(P = < 0.005), while Rt was significantly higher in termpregnancies than in either early pregnancy (P < 0.01) orthe menstrual cycle (P < 0.001). Corpora lutea atretica (n= 2) and ovarian stroma (n = 6) did not show any EGF bindingactivity. Our findings demonstrate the presence of specificEGF receptors in human corpora lutea of both the menstrual cycleand pregnancy. The changes in EGF binding parameters in earlypregnancy suggest that there may be a relationship between therole of EGF and ovarian steroidogenesis.     

Uterine natural killer cells and angiogenesis in recurrent reproductive failure

BACKGROUND: Increased numbers of phenotypically unusual CD56^bright CD16–uterine natural killer (uNK) cells have been associated withrecurrent reproductive failure. uNK cells produce angiogenicgrowth factors and are potential regulators of decidual angiogenesisin early pregnancy. The final common mechanism for early pregnancyloss is thought to be early onset of the maternal circulationand excessive placental oxidative stress. We tested the hypothesisthat increased uNK cells in preimplantation endometrium areassociated with altered angiogenesis. METHODS: Women with recurrent reproductive failure (n = 122) were investigatedwith uterine artery Doppler and endometrial biopsy. Immunohistochemistrywas used to identify uNK, endothelial and vascular smooth musclecells and image analysis was used to assess location, densityand differentiation. RESULTS: uNK cell density was positively correlated with the formationof blood (P = 0.005, r = 0.5) and lymphatic vessels (P = 0.0001,r = 0.6), spiral arteriole smooth muscle differentiation (P= 0.01, r = 0.5) and endometrial oedema (P = 0.004). The functionaleffect of this was a reduced uterine artery resistance to bloodflow. CONCLUSIONS: These data suggest that uNK cells may regulate angiogenesisin non-pregnant endometrium. The mechanisms of reproductivefailure associated with increased uNK cell density appear tobe increased angiogenesis and peri-implantation blood flow,which may lead to early maternal circulation and hence pregnancyfailure due to excessive oxidative stress.     

CD4+Thy1- thymocytes with a Th-type 2 cytokine response

We have identified a small subset of CD3⁺, CD4⁺, CD8^–thymocytes that do not express Thy1 (CD90). This Thy1^–subset represents 1–3.7% of the total number of thymocytesin a naive mouse. CD4⁺Thy1^– thymocytes express high levelsof CD3, intermediate to high levels of heat-stable antigen (HSA),and low levels of CD25, CD45RB, CD69, CD44 and CD62L. They producehigh titers of IL-4 and no IFN- upon stimulation in vitro, aresponse characteristic of T_h2 cells. In the thymi of mice infectedneonatally with a high dose of the retrovirus Cas-Br-E MuLV,the frequency of CD4⁺Thy1^– cells increased ~10-fold. High-dosevirus infection resulted in decreased HSA and increased CD44expression on CD4⁺Thy1^– cells relative to cells from naivemice. CD4⁺Thy1^– cells from high-dose infected mice alsosecreted IL-4 and not IFN- upon in vitro stimulation. We previouslyreported that infection of newborn mice with a high dose ofmurine retrovirus results in the induction of a non-protectiveanti-viral T_h2 T cell response; CD4⁺Thy1^– thymocytes witha T_h2-like cytokine profile may play a role in determining thecytokine bias of this anti-viral response.     

Andrology: Assessment of the acrosomal status and viability of human spermatozoa simultaneously using flow cytometry

Acrosomal status and viability were evaluated simultaneouslyon human spermatozoa using flow cytometry. Samples were dividedinto three aliquots and randomly assigned to one of three treatments:(i) cryopreservation; (ii) 10 µM calcium ionophore [A23187in dimethylsulphoxide (DMSO)] or (iii) DMSO alone (control).Acrosomal status was evaluated using monoclonal antibodies recognizingMH61 and CD46, respectively. Fluorescein-conjugated goat anti-mouseimmunoglobulin (IgG) was used as a second antibody. Sperm viabilitywas assessed using Hoechst 33258 (H258) exclusion. The followingfactors were analysed: (i) the specificity of the monoclonalantibodies for the human acrosome; (ii) the relative effectivenessof flow cytometry and direct fluorescent microscopy scoringand (iii) the acrosomal status and viability of the control,ionophore-treated, and cryopreserved spermatozoa. Across alltreatments, the MH61 and CD46 monoclonal antibodies resultedin acrosomal status values (acrosome-reacted/viable spermatozoa)which were not significantly different (P > 0.05): control,1.0 ± 0.3% and 1.5 ± 0.6% (mean ± SEM);A23187, 42.8 ± 3.5% and 38.1 ± 3.5%; cryopreserved,8.2 ± 2.0% and 9.9 ± 1.3%; respectively. However,acrosomal status among treatments differed significantly (P< 0.01). Flow cytometric and direct fluorescent microscopyassessments were significantly correlated (r² = 0.96, P <0.01). These results indicate that flow cytometry, using anacrosome-specific monoclonal antibody and a supravital dye,provides an objective and efficient method to evaluate humansperm acrosomal and viability status simultaneously.     

Angiogenesis in triple-negative adenoid cystic carcinomas of the breast

We compared microvascular density (MVD), lymph vessel density (LVD), and the expression of hypoxia pathway-associated proteins between primary triple-negative adenoid cystic carcinoma of the breast (TN-ACC) and grade-matched triple-negative breast carcinomas of no special type (TNBC). Twelve TN-ACC and 15 TNBC were investigated immunohistochemically for CD31, podoplanin (D2-40), von Hippel–Lindau protein (pVHL), and hypoxia-inducible factor-1alpha (HIF-1α) protein. All cases were lymph node negative (pN0). The study revealed a median MVD (CD31) of 34 vessels/mm² (mean ± SD, 41.33 ± 6.5/mm²) in the TN-ACC subgroup and a median of 55 microvessels (mean ± SD, 54.9 ± 6.3/mm²) in the TNBC subgroup. The median LVD (D2-40) was 10.5/mm² (mean ± SD, 11.9 ± 1.5/mm²) in the TN-ACC subgroup and 15.0/mm² (mean ± SD, 16.9 ± 2.5/mm²) lymph vessels in the TNBC subgroup. The differences were not statistically significant (P = 0.93, P = 0.67, respectively). pVHL was detectable in all TN-ACCs whereas two cases of TNBC had less than 5% of the positive cells. HIF-1α protein expression was significantly higher in the tumor cell population than in adjacent normal cells in both subgroups (P = 0.009 for TNBC and P = 0.028 for TN-ACC, respectively), but there was no significant difference between the two tumor groups. Up-regulation of the hypoxia-induced signaling is seen in both TN-ACC and grade-matched TNBC. Despite its perceived low malignant potential, TN-ACC of the breast does not differ in the number of blood and lymphatic vessels in comparison with the grade-matched TNBC. The reported biologic differences between TN-ACC and TNBC do not appear to result from neoangiogenesis.     

Induction of CD5 antigen on human CD5 B cells by stimulation with Staphylococcus aureus Cowan strain I

We have examined whether the CD5 phenotype could be inducedon human B cell surfaces by the polycional B cell stimulator,Staphyiococcus aureus Cowan strain I (SAC). Fresh tonsillarB cells were prepared by Percoll density gradient from E^–cells. The proportion of CD5⁺ B cells In the 50/60% and 60/70%interface high-density fractions varied between 1.2 and 10.2%depending on the tonsil preparations when they were placed onthe in vitro culture 12–60 h prior to flow cytometrlcanalysis. The expression of CD5 antigen obviously increasedin the presence of SAC (1:10⁵ v/v). The percentage of CD5⁺ Bcells varied from tonsil to tonsil, from 25.1 to 65.9% in aseries of experiments. The CD5⁺ B cells were found both amongCD23⁺CD25⁺CD71⁺ and CD23^–CD25^–CD71^– B cells.The level of CD5 expression was related to the cell size eniargement.The addition of anti-CD5 antibody in the culture blocked theCD5 induction by SAC without interfering with the expressionof other activation markers. A time-course study showed thatCD5 antigen appeared to be induced on the cell surface duringthe G₀ to G₁ phase transition in the cell cycle. When CD5⁺ andCD5^– B cells were separated by magnetic isolation, theCD5^– B cells showed DNA synthesis to the stimulation bySAC and expressed CD5 antigen on their cell surface. These resultssuggest that human CD5^– B cells can express the CD5 phenotypeby stimulationwith the polyclonal B cell stimulator, SAC.     

Progression of T cell lineage restriction in the earliest subpopulation of murine adult thymus visualized by the expression of lck proximal promoter activity

The proximal promoter of lck directs gene expression exclusivelyin T cells. To investigate the developmental regulation of thelck proximal promoter activity and its relationship to T celllineage commitment, a green fluorescence protein (GFP) transgenic(Tg) mouse in which the GFP expression is under the controlof the proximal promoter of lck was created. In the adult GFP-Tgmice, >90% of CD4⁺CD8⁺ and CD4⁺CD8^– thymocytes, andthe majority of CD4^–CD8⁺ and CD4^–CD8^– [double-negative(DN)] thymocytes were highly positive for GFP. Slightly lowerbut substantial levels of expression of GFP was also observedin mature splenic T cells. No GFP⁺ cells was detected in non-Tlineage subsets, including mature and immature B cells, CD5⁺B cells, and NK cells, indicating a preserved tissue specificityof the promoter. The earliest GFP⁺ cells detected were foundin the CD44⁺CD25^– DN thymocyte subpopulation. The developmentalpotential of GFP^– and GFP⁺ cells in the CD44⁺CD25^–DN fraction was examined using in vitro culture systems. Thegeneration of substantial numbers of ß and T cells aswell as NK cells was demonstrated from both GFP^– and GFP⁺cells. However, no development of B cells or dendritic cellswas detected from GFP⁺ CD44⁺CD25^– DN thymocytes. Theseresults suggest that the progenitors expressing lck proximalpromoter activity in the CD44⁺CD25^– DN thymocyte subsethave lost most of the progenitor potential for the B and dendriticcell lineage. Thus, progression of T cell lineage restrictionin the earliest thymic population can be visualized by lck proximalpromoter activity, suggesting a potential role of Lck in theT cell lineage commitment.     

Superantigen-reactive T cells that display an anergic phenotype in vitro appear functional in vivo

Clonal deletion and/or inactivation establishes tolerance toself antigens. Endogenous and exogenous (bacterial) superantigens,like the staphylococcal enterotoxlns, induce ligand-specificclonal anergy in vivo and thus are believed to mirror aspectsof post-thymic tolerance mechanisms in mature peripheral T cells.Here we analyzed the level of anergy of ligand-responsive V_ß8⁺T cells from staphylococcal enterotoxin B (SEB)-primed micein vivo and in vitro. Upon in vitro restimulation with SEB,CD4⁺V_ß8⁺ and CD8⁺V_ß8⁺ T cells failed toproduce IL-2. However, functional IL-2 receptors were triggered,since supplementation with IL-2 induced clonal growth in virtuallyall CD4⁺V_ß8⁺ and CD8⁺V_ß8⁺ T cells as determinedby limiting dilution analyses. Thus in vitro unresponslvenessof lymphocytes from SEB-primed mice reflects the inability ofSEB-reactlve V_ß8⁺ T cells to produce IL-2. Surprisingly,anergy as defined in vitro was at variance with that in vivo.Following further challenge with SEB, systemic and acute lymphokineproduction (Including IL-2 and tumor necrosis factor) occurredwith almost identical peak values and kinetics to primary invivo responses, and D-galactosamlne-sensltlzed mice succumbedto lethal shock. Polymerase chain reaction analyses revealedthat CD4⁺V_ß8⁺ expressed IL-2-specific mRNA in vivoupon restimulatlon with SEB. While lymphokine production andexpression of the IL-2 receptor was similar to the responseto in vivo primary stimulation, only CD8⁺V_ß8⁺ T cellsexpanded clonally upon reintroductlon of SEB in vivo. Henceprimed V_ß8⁺ T cells challenged with SEB display invitro anergy yet in vivo responsiveness, at least in part. Weconclude that the state of anergy is reversible, dependent uponthe quality of activation signals provided in in vivo ratherthan in in vitro culture conditions.     

CD4+ICOS+ T lymphocytes inhibit T cell activation 'in vitro' and attenuate autoimmune encephalitis 'in vivo'

The inducible co-stimulator (ICOS, CD278) is essential to theefficient development of normal and pathological immune reactions.Since ICOS-deficient mice have enhanced susceptibility to experimentalallergic encephalomyelitis (EAE), we have functionally analyzeda CD4⁺ICOS⁺ population comprising 6–15% of all CD4⁺ Tcells in secondary lymphoid organs of unmanipulated wild-typemice and checked for their ability to suppress EAE. In C57BL/6mice, CD4⁺ICOS⁺ cells were a major source of cytokines includingIFN-, IL-2, IL-4, IL-10 or IL-17A. Upon activation, these cellsshowed preferentially enhanced production of IL-4 or IL-10 butinhibited IFN- production. In contrast, CD4⁺ICOS^– cellsmainly produced IFN-. Interestingly, CD4⁺ICOS⁺ cells partiallysuppressed the proliferation of CD4⁺ICOS^– or CD4⁺CD25^–lymphocytes ‘in vitro’ by an IL-10-dependent mechanism.Furthermore, CD4⁺ICOS⁺ activated and expanded under appropriateconditions yielded a population enriched in cells producingIL-10 and T_h2 cytokines that also suppressed the proliferationof CD4⁺CD25^– lymphocytes. CD4⁺ICOS⁺ cells, before or afterexpansion in vitro, reduced the severity of EAE when transferredto ICOS-deficient mice. In the same EAE model, lymph node cellsfrom ICOS-deficient mice receiving ICOS⁺ cells showed reducedIL-17A production and enhanced IL-10 secretion upon antigenactivation in vitro. Thus, naturally occurring CD4⁺ICOS⁺ cells,expanded or not in vitro, are functionally relevant cells ableof protecting ICOS-deficient mice from severe EAE.     

Thyroid autoantibodies in euthyroid non-pregnant women with recurrent spontaneous abortions

This study was undertaken to evaluate the incidence of thyroidautoantibodies in women with a history of recurrent (three ormore consecutive) spontaneous abortions. A total of 22 euthyroidnon-pregnant habitual aborters were analysed for thyreoglobulinand thyroid peroxidase antibodies; 22 nulligravidae and 22 multigravidaewithout endocrine dysfunction served as controls. Both thyroidantibodies were assayed using highly sensitive, commerciallyavailable enzyme-linked immunosorbent assay kits. Eight of the22 women with recurrent spontaneous abortions (36%) but onlytwo of the 22 nulligravidae controls (9%; ² test, P = 0.03)and one of 22 multigravidae subjects (5%; ² test, P < 0.01)demonstrated positive titres (> 100 IU/ml, as recommendedby the manufacturer) of thyreoglobulin, thyroid peroxidase orboth antibodies. The mean ± SD antibody concentrationswere 102.5 ± 226.5 in the study versus 20.9 ±68.8 in the nulligravidae (U-test, P = 0.057) and 26.4 ±60.2 IU/ml (P = 0.097) in the multigravidae population for thyroidperoxidase, and 47.7 ± 57.9 versus 24.1 ± 31.9(U-test, P = 0.051) and 28.1 ± 27.9 IU/ml (P = 0.059)for thyreoglobulin. In conclusion, the incidence of thyroidantibodies in euthyroid women with habitual abortions appearsto be significantly increased compared with the controls ofreproductive age without previous abortions. Although the importantissue of cause and effect has not been fully clarified, thisfinding suggests that thyroid antibodies may be a marker forautoimmune-mediated recurrent spontaneous abortions, not dissimilarto, for example, anticardiolipin.     

Vaccination with DNA encoding internal proteins of influenza virus does not require CD8(+) cytotoxic T lymphocytes: either CD4(+) or CD8(+) T cells can promote survival and recovery after challenge

DNA vaccination offers the advantages of viral gene expressionwithin host cells without the risks of infectious virus. Likeviral vaccines, DNA vaccines encoding internal influenza virusproteins can induce immunity to conserved epitopes and so maydefend the host against a broad range of viral variants. CD8⁺cytotoxic T lymphocytes (CTL) have been described as essentialeffectors in protection by influenza nucleoprotein (NP), althougha lesser role of CD4⁺ cells has been reported. We immunizedmice with plasmids encoding influenza virus NP and matrix (M).NP + M DNA allowed B6 mice to survive otherwise lethal challengeinfection, but did not protect B6-ß₂m(–/–)mice defective in CD8⁺ CTL. However, this does not prove CTLare required, because ß₂m(–/–) mice have multipleimmune abnormalities. We used acute T cell depletion in vivoto identify effectors critical for defense against challengeinfection. Since lung lymphocytes are relevant to virus clearance,surface phenotypes and cytolytic activity of lung lymphocyteswere analyzed in depleted animals, along with lethal challengestudies. Depletion of either CD4⁺ or CD8⁺ T cells in NP + MDNA-immunized BALB/c mice during the challenge period did notsignificantly decrease survival, while simultaneous depletionof CD4⁺ and CD8⁺ cells or depletion of all CD90⁺ cells completelyabrogated survival. We conclude that T cell immunity inducedby NP + M DNA vaccination is responsible for immune defense,but CD8⁺ T cells are not essential in the active response tothis vaccination. Either CD4⁺ or CD8⁺ T cells can promote survivaland recovery in the absence of the other subset.     

Endocrinology: The induction of ovulation with pulsatile gonadotrophin-releasing hormone (GnRH) administration in hyperandrogenic women after down-regulation with buserelin or suppression with an oral contraceptive

The hypothalamic—pituitary axis of 22 hyperandrogenicinfertile women had suppression with either the gonadotrophin-releasinghormone (GnRH)-analogue buserelin (n = 12) or with an oestrogen—gestagencompound (Diane^®; ⁿ = 12). This was followed by pulsatileGnRH application for inducing ovulation (Zyklomat^®). Interms of ovulation and pregnancy rates the buserelin pre-treatmentwas more effective than the steroid pre-treatment, especiallyin hyperandrogenic non-polycystic ovaries (PCO).