Haematological Changes During Normal Pregnancy in New Zealand White Rabbits: A Longitudinal Study

The present study was undertaken to investigate changes in haematology parameters over the course of normal pregnancy in New Zealand White rabbits. Blood samples were collected on gestational days (GD) 0, 4, 8, 12, 16, 20, 24, and 28. Red blood cell counts and haemoglobin concentrations on GD 20–28 were lower than those of normal non-pregnant rabbits. These values fluctuated slightly between GD 0 and 12 and subsequently decreased to reach a nadir on either GD 24 or 28. Haematocrit value in pregnant rabbits also decreased slightly in the third trimester, but the difference was not statistically noticeable. Mean corpuscular volume in pregnant rabbits increased gradually during the course of gestation and was larger on GD 24 than that in non-pregnant rabbits. There were no differences in mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration between pregnant and non-pregnant rabbits. Platelet counts on GD 24–28 were lower than that of normal non-pregnant rabbits. These values increased slightly in the first half of gestation and then decreased to reach a nadir on GD 28. Total white blood cell and lymphocyte counts on GD 24 were lower than those of normal non-pregnant rabbits. These values increased maximally by GD 4 and then decreased progressively to a minimum level on GD 24. No significant differences were observed in the numbers of neutrophils, eosinophils, basophils, and monocytes between pregnant and non-pregnant rabbits. Neutrophil counts of pregnant rabbits fluctuated minimally between GD 0 and 12 and then decreased to reach a lowest level on GD 24. Eosinophil counts increased to a maximum value on GD 4 and subsequently decreased to reach a nadir on GD 24. Basophil and monocyte counts were not different throughout the course of pregnancy. These data can be used not only as a historical database for the effective evaluation of data from reproductive toxicology studies, but also as a contribution to biological characterization of New Zealand White rabbits.     

In Vivo Tumour Mapping Using Electrocorticography Alterations During Awake Brain Surgery: A Pilot Study

During awake brain surgery for tumour resection, in situ EEG recording (ECoG) is used to identify eloquent areas surrounding the tumour. We used the ECoG setup to record the electrical activity of cortical and subcortical tumours and then performed frequency and connectivity analyses in order to identify ECoG impairments and map tumours. We selected 16 patients with cortical (8) and subcortical (8) tumours undergoing awake brain surgery. For each patient, we computed the spectral content of tumoural and healthy areas in each frequency band. We computed connectivity of each electrode using connectivity markers (linear and non-linear correlations, phase-locking and coherence). We performed comparisons between healthy and tumour electrodes. The ECoG alterations were used to implement automated classification of the electrodes using clustering or neural network algorithms. ECoG alterations were used to image cortical tumours.Cortical tumours were found to profoundly alter all frequency contents (normalized and absolute power), with an increase in the δ activity and a decreases for the other bands (P < 0.05). Cortical tumour electrodes showed high level of connectivity compared to surrounding electrodes (all markers, P < 0.05). For subcortical tumours, a relative decrease in the γ1 band and in the alpha band in absolute amplitude (P < 0.05) were the only abnormalities. The neural network algorithm classification had a good performance: 93.6 % of the electrodes were classified adequately on a test subject. We found significant spectral and connectivity ECoG changes for cortical tumours, which allowed tumour recognition. Artificial neural algorithm pattern recognition seems promising for electrode classification in awake tumour surgery.     

In Vitro and In Vivo Interactions of Haemophilus ducreyi with Host Phagocytes

We investigated the phagocytosis of Haemophilus ducreyi both in vitro and in vivo. Human granulocyte and monocyte phagocytosis of opsonized and nonopsonized, fluorescence-labeled H. ducreyi was assessed by flow cytometry. Both Escherichia coli and noncapsulated H. influenzae were included as controls. The maximal percentage of granulocytes taken up by H. ducreyi was 35% after 90 min. In contrast, 95% of H. influenzae bacteria were phagocytosed by granulocytes after 30 min. These results indicated that H. ducreyi phagocytosis was slow and inefficient. Bacterial opsonization by using specific antibodies increased the percentage of granulocytes phagocytosing H. ducreyi from 24 to 49%. The nonphagocytosed bacteria were completely resistant to phagocytosis even when reexposed to granulocytes, indicating that the H. ducreyi culture comprised a mixture of phenotypes. The intracellular survival of H. ducreyi in granulocytes, in monocytes/macrophages, and in a monocyte cell line (THP-1) was quantified after application of gentamicin treatment to kill extracellular bacteria. H. ducreyi survival within phagocytes was poor; approximately 11 and <0.1% of the added bacteria survived intracellularly after 2 and 20 h of incubation, respectively, while no intracellular H. influenzae bacteria were recovered after 2 h of incubation with phagocytes. The role of phagocytes in the development of skin lesions due to H. ducreyi was also studied in vivo. Mice that were depleted of granulocytes and/or monocytes and SCID mice, which lacked T and B cells, were injected intradermally with approximately 10⁶ CFU of H. ducreyi. Within 4 days of inoculation, the granulocyte-depleted mice developed lesions that persisted throughout the experimental period. This result reinforces the importance of granulocytes in the early innate defense against H. ducreyi infection. In conclusion, H. ducreyi is insufficiently phagocytosed to achieve complete eradication of the bacteria. Indeed, H. ducreyi has the ability to survive intracellularly for short periods within phagocytic cells in vitro. Since granulocytes play a major role in the innate defense against H. ducreyi infection in vivo, bacterial resistance to phagocytosis probably plays a crucial role in the pathogenesis of chancroid.     

自然步态摆动期动力学协调模式的研究

建立了包含肌肉一肌腱动力特性和神经兴奋一肌肉收缩动力学方程在内的人体下肢生物力学模型，并针对自然步态摆动期的运动过程，采用最优控制方法进行了计算分析。计算结果与实验测量结果在运动学、肌电信号和肌肉活性参数等方面均具有很好的一致性；同时利用模型分析了肌肉协调运动模式，结果表明在自然步态摆动期运动过程中，神经系统将肌肉结成不同的协同肌群，并按照特定的时序结构控制不同肌群实现步行运动的加速、制动和姿态控制。     

Estimation of Ligament Loading and Anterior Tibial Translation in Healthy and ACL-Deficient Knees During Gait and the Influence of Increasing Tibial Slope Using EMG-Driven Approach

The purpose of this study was to develop a biomechanical model to estimate anterior tibial translation (ATT), anterior shear forces, and ligament loading in the healthy and anterior cruciate ligament (ACL)-deficient knee joint during gait. This model used electromyography (EMG), joint position, and force plate data as inputs to calculate ligament loading during stance phase. First, an EMG-driven model was used to calculate forces for the major muscles crossing the knee joint. The calculated muscle forces were used as inputs to a knee model that incorporated a knee–ligament model in order to solve for ATT and ligament forces. The model took advantage of using EMGs as inputs, and could account for the abnormal muscle activation patterns of ACL-deficient gait. We validated our model by comparing the calculated results with previous in vitro, in vivo, and numerical studies of healthy and ACL-deficient knees, and this gave us confidence on the accuracy of our model calculations. Our model predicted that ATT increased throughout stance phase for the ACL-deficient knee compared with the healthy knee. The medial collateral ligament functioned as the main passive restraint to anterior shear force in the ACL-deficient knee. Although strong co-contraction of knee flexors was found to help restrain ATT in the ACL-deficient knee, it did not counteract the effect of ACL rupture. Posterior inclination angle of the tibial plateau was found to be a crucial parameter in determining knee mechanics, and increasing the tibial slope inclination in our model would increase the resulting ATT and ligament forces in both healthy and ACL-deficient knees.     

In Vivo Elimination of Allogeneic Lymphocytes in Normal and T-Cell-Deficient Rats

The distribution of allogeneic and syngeneic thoracic duct lymphocytes was studied over 24 h in normal and T-cell-deficient animals (thymectomized, irradiated rats, 'B rats', or congenitally athymic nude rats). Initial migration from blood was no less for allogenic than for syngeneic cells. After 24 h, however, a marked deficit of radioactively labelled allogeneic cells as compared with syngeneic cells was found in the lymphoid tissue, whereas the allogeneic isotope was recovered in a relatively greater amount in liver, kidneys, and cell-free plasma and lymph. Most of the allogeneic cells are evidently destroyed within the first 24 h and their isotope released into body fluids. Our studies also revealed this process to be even more evident in T-cell-depleted environments. Autoradiographic studies of recipient nude rat spleens showed that allogeneic cells were not found in the great number seen in syngeneic transfers, but a high grain density in the periarteriolar lymphocyte sheath area could be observed. Granula seemed to be predominantly located over large nonlymphoid cells. The elimination of allogeneic lymphocytes is therefore governed by mechanisms independent of an intact thymus and may be due to a cell population or factor more active in nude animals than in their non-nude littermates.     

The Role of Macrophages in Utero-placental Interactions During Normal and Pathological Pregnancy

The intimate association between maternal and placental tissues elicits an interesting immunological paradox. Placental tissue contains paternal antigens, but under normal circumstances the semi-allogeneic fetus and placenta are not attacked by the maternal immune system. Interestingly, this tolerance to fetal antigens occurs in the presence of a large number of maternal leukocytes, almost all of which are members of the innate immune system. Macrophages are one of the most abundant leukocytes in the decidua and their numbers remain constant throughout gestation. They are recruited to the decidua by both stromal cells and trophoblast cells, where they adopt a specialized phenotype that may assist in various aspects of decidual homeostasis, placental development, and tolerance to the semi-allogeneic trophoblast. Aberrant behavior of these macrophages can affect trophoblast function and placental development, potentially leading to a spectrum of adverse pregnancy outcomes ranging from pre-eclampsia to fetal growth restriction or demise. This review will focus on the phenotype and putative functions of decidual macrophages in normal pregnancy, and how abnormal activation of these cells can affect various aspects of placental development.     

Differentiation of Normal and Malignant Human Squamous Epithelium in Vivo and in Vitro: A Morphologic Study

We would like to thank John Ellis for expert photographic assistance, Mervin Jones and Linda Lovell for expert animal husbandry, and Dr. Dorothy Easty for establishing the cell line LICR-LON-HN-5, without which this study would not have been possible.

We report a light microscopic and ultrastructural analysis of the comparative degrees of differentiation seen in keratinocytes derived from the tongue and epidermis with those of a well-differentiated human squamous carcinoma cell line (LICR-LON-HN5). When growing on plastic substrates, all cultures had a similar morphology, with multilayering and the production of cornified envelopes. When cultured on collagen gels the structure was more organized, with keratohyalin granules and keratin whorl formation in both the normal and the malignant cultures. Normal keratinocytes injected into athymic mice produced epidermal cysts, while cells from the cell line produced well-differentiated squamous cell carcinomas, which were partially solid and partially cystic. The tumor was well organized, with identifiable basal cells, spin-ous cells, keratohyalin granules, and a prominent basal lamina at the stromal/epithelial interface. This model is to be developed for comparative studies between normal and malignant cells, with particular reference to basement membrane production and to investigations of the relative importance of extrinsic and intrinsic factors in the control of squamous differentiation.     

Differentiation of Normal and Malignant Human Squamous Epithelium in Vivo and in Vitro: A Morphologic Study

We would like to thank John Ellis for expert photographic assistance, Mervin Jones and Linda Lovell for expert animal husbandry, and Dr. Dorothy Easty for establishing the cell line LICR-LON-HN-5, without which this study would not have been possible.

We report a light microscopic and ultrastructural analysis of the comparative degrees of differentiation seen in keratinocytes derived from the tongue and epidermis with those of a well-differentiated human squamous carcinoma cell line (LICR-LON-HN5). When growing on plastic substrates, all cultures had a similar morphology, with multilayering and the production of cornified envelopes. When cultured on collagen gels the structure was more organized, with keratohyalin granules and keratin whorl formation in both the normal and the malignant cultures. Normal keratinocytes injected into athymic mice produced epidermal cysts, while cells from the cell line produced well-differentiated squamous cell carcinomas, which were partially solid and partially cystic. The tumor was well organized, with identifiable basal cells, spin-ous cells, keratohyalin granules, and a prominent basal lamina at the stromal/epithelial interface. This model is to be developed for comparative studies between normal and malignant cells, with particular reference to basement membrane production and to investigations of the relative importance of extrinsic and intrinsic factors in the control of squamous differentiation.     

T and B Lymphocytes during Normal Human Pregnancy: a Longitudinal Study

Immunological relationships in pregnancy were investigated in a longitudinal study of twenty-two pregnant women, whose blood samples were taken before pregnancy, during pregnancy, at delivery, and 3--5 months after delivery. Blood samples were also taken from the fathers and the infants, both at birth and after 3--5 months. All the samples were frozen by means of a cryobiological freezing system, and when a whole longtiudinal series had been collected, the material was thawed and tested in a single seance. T and B lymphocytes were studied with rosette tests (E and HEAC rosettes). T and B lymphocytes were found not to change during the course of pregnancy. It is thus concluded that the mother's tolerance of a fetus with a dissimilar tissue type is not exercised via changes in the total count of T and B lymphocytes, although there may well be changes in their subpopulations, with the hypothesis that T-suppressor function increases and B-lymphocyte function decreases.     

Functional Activity of the Anterior and Posterior Cruciate Ligaments Under In Vivo Gait and Static Physiological Loads

The loading of the anterior and posterior cruciate ligaments (ACL and PCL) during normal gait has not been quantified. Also, it is not clear whether ligaments under “static” physiological loads commonly used in cadaveric studies behave similarly to normal gait experienced in vivo. We measured the in vivo kinematics of the stifle joint of sheep (N = 4) during “normal gait,” then reproduced these gait paths using a robotic system. The loads borne by the cruciate ligaments were determined using the principle of superposition and plotted against each other. This indicated some functional interaction between the ACL and PCL under in vivo physiological loads. To examine this relationship under static loading conditions, cadaveric knees (N = 6) were tested in the anterior–posterior (AP) direction, along the axes of the ACL and the PCL, as well as under combined AP and tibial rotations. The same process was repeated after either the ACL (N = 3) or the PCL (N = 3) was transected. Our results show a mutually exclusive relationship in ACL and PCL load bearing under both “in vivo gait” and “static” loading conditions. High ACL loads were associated with low PCL loads and vice versa. This is a novel study quantifying the actions of the cruciate ligaments during gait and comparing them to commonly used static loading conditions in cadaveric studies.     

Longitudinal Memory Performance During Normal Aging: Twin Association Models of APOE and Other Alzheimer Candidate Genes

The APOE gene (apolipoprotein E) is a major risk factor for Alzheimer’s Disease (AD) but has been inconsistently associated with memory in nondemented adults. Two other genes with mixed support as genetic risk factors for AD, A2M (alpha-2-macroglobulin) and LRP (low-density lipoprotein receptor-related protein), have not been studied in relation to memory among nondemented adults. The present study examined these three genes and latent growth parameters estimated from memory performance spanning 13 years in 478 twins from the Swedish Adoption/Twin Study of Aging (SATSA). APOE was associated with working and recall memory ability levels and working memory rate of change, with e4 homozygotes exhibiting the worst performance at all ages. Homozygotes for the rare A2M insertion/deletion variant exhibited accelerating decline on delayed figural recognition. There were no significant findings for LRP. Dominance, often untested in previous studies, was important in the current study’s findings.     

兔眼前房压强在体连续测量方法的研究

为了解眼睛房水压强变化规律,进一步建立闭角型青光眼模型和对疾病的治疗及视功能康复提供参考,探讨一种在体连续测量正常兔眼前房压强的方法。将正常成年新西兰白兔麻醉后,先用静脉留置针(20G)自角膜缘外行前房穿刺术,然后通过套管将Millar导管植入兔眼的前房,兔眼的前房压强信号经过Powerlab系统进行滤波、放大和模数转换后,通过Chart软件进行实时显示。结果表明:用该方法测得的正常兔眼24 h前房压强的变化范围为(1.31±0.21—2.74±0.83)KPa。前房压强的分布凌晨较低(0~5点),然后逐渐升高(5~10点),到中午或下午(11—17点)时达到最高峰,晚上又逐渐下降(18~23点)。结果证明本实验在体连续测量兔眼前房压强的方法是可行的。     

In Vivo Demyelination Induced by Intraneural Injection of Anti-Galactocerebroside Serum: A Morphologic Study

Intraneural injection of rabbit anti-galactocerebroside (anti-GC) serum produced focaldemyelinative lesions in rat sciatic nerves. Recipient rats developed a sensory motordeficit of the toes and feet on the side injected with anti-GC serum. Schwann cellabnormalities in recipient nerves were apparent by 20 minutes, followed by myelinsplitting and vesiculation over the next 8 hours. Macrophages first appeared in moder-ate numbers by 15 hours, and degraded myelin was completely phagocvtized by 5 days.An acute inflammatory reaction consisting of endoneurial edema, polymorphonuclearcell infiltration, and fibrin extravasation also was prominent. In vivo demyelinativeactivity of rabbit anti-GC serums was removed by pre-incubation with GC or central orperipheral nervous system myelin and was also lost when the serums were heated at 56C for 30 minutes and injected into nerves of rats previously injected with cobra venomfactor. Anti-GC antibodies are present in the serum of rabbits with experimentalallergic neuritis (WNV-EAN) and encephalomyelitis (WM-EAE) produced, respectively,by immunization with whole peripheral nerve or brain white matter and may play arole in the pathogenesis of demyelination in GC-induced EAN, WN-EAN, or WM-EAE.     

Immunomodulatory Effects of Tyrosine Kinase Inhibitor In Vitro and In Vivo Study

Pathogenesis of chronic graft-versus-host disease (cGVHD) is incompletely defined, involving donor-derived CD4 and CD8-positive T lymphocytes as well as B cells. Standard treatment is lacking for steroid-dependent/refractory cases; therefore, the potential usefulness of tyrosine kinase inhibitors (TKIs) has been suggested, based on their potent antifibrotic effect. However, TKIs seem to have pleiotropic activity. We sought to evaluate the in vitro and in vivo impact of different TKIs on lymphocyte phenotype and function. Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured in the presence of increasing concentrations of nilotinib, imatinib, dasatinib, and ponatinib; in parallel, 44 PBMC samples from 15 patients with steroid-dependent/refractory cGVHD treated with nilotinib in the setting of a phase I/II trial were analyzed at baseline, after 90, and after 180 days of therapy. Flow cytometry was performed after labeling lymphocytes with a panel of monoclonal antibodies (CD3, CD4, CD16, CD56, CD25, CD19, CD45RA, FoxP3, CD127, and 7-amino actinomycin D). Cytokine production was assessed in supernatants of purified CD3⁺?T cells and in plasma samples from nilotinib-treated patients. Main T lymphocyte subpopulations were not significantly affected by therapeutic concentrations of TKIs in vitro, whereas proinflammatory cytokine (in particular, IL-2, IFN-γ, tumor necrosis factor-α, and IL-10) and IL-17 production showed a sharp decline. Frequency of T regulatory, B, and natural killer (NK) cells decreased progressively in presence of therapeutic concentrations of all TKIs tested in vitro, except for nilotinib, which showed little effect on these subsets. Of note, naive T regulatory cell (Treg) subset accumulated after exposure to TKIs. Results obtained in vivo on nilotinib-treated patients were largely comparable, both on lymphocyte subset kinetics and on cytokine production by CD3-positive cells. This study underlines the anti-inflammatory and immunomodulatory effects of TKIs and supports their potential usefulness as treatment for patients with steroid-dependent/refractory cGVHD. In addition, both in vitro and in vivo data point out that compared with other TKIs, nilotinib could better preserve the integrity of some important regulatory subsets, such as Treg and NK cells.     

A Poly(lactic-co-glycolic acid) Knitted Scaffold for Tendon Tissue Engineering: An In Vitro and In Vivo Study

We have designed a composite scaffold for potential use in tendon or ligament tissue engineering. The composite scaffold was made of a cellularized alginate gel that encapsulated a knitted structure. Our hypothesis was that the alginate would act as a cell carrier and deliver cells to the injury site while the knitted structure would provide mechanical strength to the composite construct. The mechanical behaviour and the degradation profile of the poly(lactic-co-glycolic acid) knitted scaffolds were evaluated. We found that our scaffolds had an elastic modulus of 750 MPa and that they lost their physical integrity within 7 weeks of in vitro incubation. Autologous rabbit mesenchymal stem cell seeded composite scaffolds were implanted in a 1-cm-long defect created in the rabbit tendon, and the biomechanical properties and the morphology of the regenerated tissues were evaluated after 13 weeks. The regenerated tendons presented higher normalized elastic modulus of (60%) when compared with naturally healed tendons (40%). The histological study showed a higher cell density and vascularization in the regenerated tendons.