Hysterosalpingography in Cynomolgus Monkeys

This paper describes recent success in performing hysterosalpingography in cynomolgus monkeys. The animals were anesthesized, and a needle was manipulated through the cervical canal into the endometrial cavity for injection of contrast material. Difficulties in threading the needle through the canal were due to several blind pouches formed by folds in the cervical mucosa. The uterine lumen and both tubes could be visualized in over 75% of the cases. The study demonstrates that the hysterosalpingography technique can be performed satisfactorily in cynomolgus monkeys.     

Improved collection and developmental competence of immature macaque oocytes

Methods previously described to aspirate immature oocytes from ovaries of macaques result in approximately half the oocytes being stripped of cumulus cells. Here, we describe modifications of the needle aspiration assembly that yield much higher percentages of cumulus-intact oocytes when used with an ultrasound-guided method for oocyte recovery in monkeys. Sealing of the needle assembly appears to stabilize vacuum pressure at the needle tip and prevents air from entering the tubing. Reduction of the vacuum pressure from -100 to -20 kPa resulted in a significant decrease of denuded oocytes from over 50% to fewer than 10%. This was accompanied by a significant increase in the percentage of oocytes that developed into blastocysts after in vitro fertilization. Reduction of the aspiration pressure below -20 kPa significantly reduced the total number of oocytes recovered. We concluded that these modifications represent the best compromise to collect the largest number of cumulus-intact oocyte complexes from macaques.     

EV71灭活疫苗（人二倍体细胞）在恒河猴婴猴模型中的免疫保护性分析

肠道病毒71型作为引起儿童群体常见传染性手足口病（HFMD）的主要病原,具有导致少量感染个体出现脑炎等神经系统病变以及相关心肺功能衰竭的病理学特性．因此其预防性疫苗的研发具有重要的公共卫生意义．在前期工作的基础上,一种EV71灭活病毒疫苗（人二倍体细胞）在本研究中基于恒河猴婴猴模型进行了相应的免疫保护性分析．以160EU剂量对2～3月龄婴猴进行0,4周免疫后,动物在第4周接受了剂量为10。～CCID50的病毒经呼吸道的攻击．对病毒攻击后动物在14天内的临床症状、血液生物学、器官病原学分布以及病理学检测的动态观察表明,经疫苗免疫的动物未出现对照动物所具有的特征性临床表现,其血液生物学及病理学检测均无异常．同时,器官病原学分布亦呈阴性．结合动物中和抗体的明确增长及对照动物的综合表现分析,本文的工作证实了该EV71灭活病毒疫苗（人二倍体细胞）在恒河猴婴猴体内的免疫保护性．     

Immune response to a hepatitis B DNA vaccine in Aotus monkeys: a comparison of vaccine formulation, route, and method of administration.

BACKGROUND: Attempts to optimize DNA vaccines in mice include using different routes of administration and different formulations. It may be more relevant to human use to carry such studies out in nonhuman primates. Here we compare different approaches to delivery of a DNA vaccine against the hepatitis B virus (HBV) in Aotus monkeys. MATERIALS AND METHODS: Thirty-two adult Aotus l. lemurinus monkeys divided into 8 groups of four were immunized with 400 microg of a DNA vaccine which encoded hepatitis B surface antigen (HBsAg). DNA in saline was administered by intradermal (ID) or intramuscular (IM) injection with needle and syringe, IM injection with the Biojector needleless injection system or combined ID (needle) and IM (Biojector). DNA formulated with cationic liposomes (CellFECTIN) was injected IM with needle or Biojector. DNA with added E. coli DNA (100 microg) was injected IM with the Biojector or ID. A ninth group of 4 monkeys was injected IM (needle) with Engerix-B, a commercial vaccine containing recombinant HBsAg (10 microg) adsorbed onto alum. Monkeys were boosted in an identical fashion to their prime at 8 weeks, but all received the protein vaccine (Engerix-B) at 16 weeks. Sera was assessed for antibodies against HBsAg (anti-HBs) by enzyme-linked imunosorbent assay (ELISA). RESULTS: The primary humoral response induced by IM delivery of the DNA vaccine was very poor. In most cases there was no detectable anti-HBs even after 2 DNA doses but the kinetics of the response to subsequent protein indicated that a memory B cell response had been induced. In contrast, following IM-administration of DNA using the Biojector, detectable anti-HBs were observed in 3 of 8 animals and evidence for immunological priming was apparent in an additional 4 of the 8 monkeys. ID injection of DNA vaccine in saline induced a potent antibody response which was augmented 6-fold by the addition of E. coli DNA. Combining ID and IM administration did not improve humoral immunity over ID injection alone. CONCLUSIONS: For immunization of primates with DNA vaccines, ID may be a preferable route to IM, although it is not clear whether the Aotus monkey is a relevant model for humans in this respect. Nevertheless, the use of the Biojector needleless injection system may improve responses with IM delivery of DNA vaccines. As well, the immunostimulatory action of E. coli DNA may be used to augment the humoral response induced by a DNA vaccine.     

Subcutaneous tissue fibroblast cytoskeletal remodeling induced by acupuncture: evidence for a mechanotransduction-based mechanism

Acupuncture needle rotation has been previously shown to cause specific mechanical stimulation of subcutaneous connective tissue. This study uses acupuncture to investigate the role of mechanotransduction-based mechanisms in mechanically-induced cytoskeletal remodeling. The effect of acupuncture needle rotation was quantified by morphometric analysis of mouse tissue explants imaged with confocal microscopy. Needle rotation induced extensive fibroblast spreading and lamellipodia formation within 30 min, measurable as an increased in cell body cross sectional area. The effect of rotation peaked with two needle revolutions and decreased with further increases in rotation. Significant effects of rotation were present throughout the tissue, indicating the presence of a response extending laterally over several centimeters. The effect of rotation with two needle revolutions was prevented by pharmacological inhibitors of actomyosin contractility (blebbistatin), Rho kinase (Y-27632 and H-1152), and Rac signaling. The active cytoskeletal response of fibroblasts demonstrated in this study constitutes an important step in understanding cellular mechanotransduction responses to externally applied mechanical stimuli in whole tissue, and supports a previously proposed model for the mechanism of acupuncture involving connective tissue mechanotransduction.     

Fetal blood sampling through ultrasound-guided puncture of the umbilical vein in rhesus monkeys

Fetal blood was obtained through puncture of the umbilical vein in Rhesus monkeys. The puncture needle was introduced during direct visual control through ultrasound. This method offers a possibility to obtain fetal blood without opening the uterine cavity. Injection into the fetal circulation is also possible.     

The anatomy of adipose tissue in captive Macaca monkeys and its implications for human biology

In a sample of 31 sedentary, ad libitum-fed monkeys, most specimens had less than 5% adipose tissue by weight. Total fatness correlated closely with the number of adipocytes per kilogram lean body mass, but not at all with mean adipocyte volume, except in specimens below 5% fat. The total number of adipocytes per kilogram of lean body mass increased more than tenfold in the most obese specimens. These data suggest that, like humans but in contrast to laboratory rodents, adipocyte proliferation, not adipocyte enlargement, is the chief mechanism of adipose tissue expansion except in very lean monkeys. Adipose tissue was found in all the typical mammalian depots and in the superficial abdominal paunch, which enlarged disproportionately in obese specimens, forming an almost continuous layer over most of the body. Site-specific differences in the activities of some glycolytic enzymes were similar to those of other mammals. Adipocytes in the paunch depot showed biochemical properties in common with those in the groin depots. The distribution and cellularity of adipose tissue in normal humans were similar to those of exceptionally obese monkeys. Many of the interspecific and sex differences can be attributed to the much greater abundance of adipose tissue in humans, and may not be associated with hair reduction or aquatic habits. Some minor changes in the size or shape of certain adipose depots may have arisen recently under sexual selection. The relevance of laboratory rodents as animal models of human obesity is assessed from comparison of the cellular structure, anatomical distribution and enzyme profiles of adipose tissue in monkeys with those of human and other mammals.     

Gamma interferon-mediated inflammation is associated with lack of protection from intravaginal simian immunodeficiency virus SIVmac239 challenge in simian-human immunodeficiency virus 89.6-immunized rhesus macaques

Although gamma interferon (IFN-gamma) is a key mediator of antiviral defenses, it is also a mediator of inflammation. As inflammation can drive lentiviral replication, we sought to determine the relationship between IFN-gamma-related host immune responses and challenge virus replication in lymphoid tissues of simian-human immunodeficiency virus 89.6 (SHIV89.6)-vaccinated and unvaccinated rhesus macaques 6 months after challenge with simian immunodeficiency virus SIVmac239. Vaccinated-protected monkeys had low tissue viral RNA (vRNA) levels, vaccinated-unprotected animals had moderate tissue vRNA levels, and unvaccinated animals had high tissue vRNA levels. The long-term challenge outcome in vaccinated monkeys was correlated with the relative balance between SIV-specific IFN-gamma T-cell responses and nonspecific IFN-gamma-driven inflammation. Vaccinated-protected monkeys had slightly increased tissue IFN-gamma mRNA levels and a high frequency of IFN-gamma-secreting T cells responding to in vitro SIVgag peptide stimulation; thus, it is likely that they could develop effective anti-SIV cytotoxic T lymphocytes in vivo. In contrast, both high tissue IFN-gamma mRNA levels and strong in vitro SIV-specific IFN-gamma T-cell responses were detected in lymphoid tissues of vaccinated-unprotected monkeys. Unvaccinated monkeys had increased tissue IFN-gamma mRNA levels but weak in vitro anti-SIV IFN-gamma T-cell responses. In addition, in lymphoid tissues of vaccinated-unprotected and unvaccinated monkeys, the increased IFN-gamma mRNA levels were associated with increased Mig/CXCL9, IP-10/CXCL10, and CXCR3 mRNA levels, suggesting that increased Mig/CXCL9 and IP-10/CXCL10 expression resulted in recruitment of CXCR3(+) activated T cells. Thus, IFN-gamma-driven inflammation promotes SIV replication in vaccinated-unprotected and unvaccinated monkeys. Unlike all unvaccinated monkeys, most monkeys vaccinated with SHIV89.6 did not develop IFN-gamma-driven inflammation, but they did develop effective antiviral CD8(+)-T-cell responses.     

Diagnosing breast carcinoma in young women.

OBJECTIVE--To assess the individual and combined diagnostic accuracy of clinical examination, mammography, and fine needle aspiration biopsy in young women with breast cancer. DESIGN--Analysis based on case notes of patients presenting with breast cancer during 1971-89. SETTING--A combined breast clinic. PATIENTS--Consecutive series of 81 women aged less than 36 with histologically proved breast cancer presenting with a discrete mass over 19 years. MAIN OUTCOME MEASURES--Results of clinical examination, xeromammography or conventional mammography, fine needle aspiration biopsy, and examination of tissue removed by surgery. RESULTS--The clinical diagnosis was correct in 47 women and radiography in 35. Fine needle aspiration biopsy was correct in 47 of the 63 women in whom it was successfully performed. Fine needle aspiration was significantly more accurate than mammography (78% v 45%, p less than 0.01). Ten (16%) patients had negative results on clinical examination, mammography, and fine needle aspiration. CONCLUSION--Mammography alone seems inadequately sensitive to detect breast cancer in young patients. When all investigations give negative results excision biopsy is the only way of obtaining a definitive diagnosis.     

Measurement of subcutaneous tissue fluid pressure using a skin-cup method

We developed a new method for measuring tissue fluid pressure in subcutaneous tissue. Porous Teflon cylinders were permanently implanted subcutaneously into the inguinal area of 10 dogs, and after several weeks a skin concavity formed in the center of each of the cylinders. A small needle attached to a recording system was inserted into the free tissue fluid lining the concavity, and the tissue fluid pressure averaged -8.8 +/- 2.7 (SD) mmHg. Next, a hollow Plexiglas cup was placed over the concavity and glued to the skin. The air pressure in the skin cup was continually adjusted (using an electromechanical servo-control system) to pull the skin upward and to hold it perfectly flat across the upper ridge of the Teflon cylinder. The simultaneously recorded needle and cup pressures averaged -9.1 +/- 2.4 and -8.6 +/- 2.6 mmHg, respectively, during steady-state conditions with the skin in a flat position. Both pressures also responded appropriately to dynamic changes in tissue fluid pressure caused by increasing and decreasing the volume of the free tissue fluid. Because the skin was flat, the equivalences of pressures above and below the skin is consistent with the hypothesis that the skin was not tethered significantly to the underlying tissues and that cup pressure accurately estimates the tissue free fluid pressure.     

Resistance forces acting on suture needles

Understanding the resistance forces encountered by a suture needle during tissue penetration is important for the development of robotic surgical devices and virtual reality surgical simulators. Tensile forces applied to skin and tendon during suturing were measured. Fresh sheep achilles tendons were tensioned with a static load 4.9 N, 9.8 N or 19.6 N and sheepskin with 0.98 N, 2.9 N or 4.9 N static load. A straight 2/0 cutting suture needle in series with a load cell on a materials testing machine penetrated the tissue at 90 degrees with a velocity of 1, 5 or 10mm/s for each tissue tension (n=5). Continuous load versus displacement data was obtained and penetration load and stiffness were noted. The load versus displacement curve for skin during needle penetration demonstrated two characteristic peaks, corresponding to initial penetration and emergence of needle from the undersurface of the tissue. Increasing the tension within the tissue (skin and tendon) increased the amount of force required to penetrate the tissue with a suture needle (p<0.05). Needle displacement rate did not affect the resistance to needle penetration (p<0.05). This study provides a simple model for measuring force-feedback during needle penetration of soft tissues and is a good starting point for future studies of the penetration resistance properties of human tissues.     

Puncture threshold prior to leakage from tissue expander reservoirs

An ex vivo study was devised to ascertain the maximum number of needle punctures possible prior to onset of leakage from remote-type tissue expansion reservoirs. At least 10 standard valves obtained from all major manufacturers were analyzed using various needle gauges. An 18-gauge beveled needle was determined to be the maximum size that could be safely employed for all vendors within a usual course for tissue expansion. No significant difference was found in comparison of Dacron-reinforced versus plain silicone membrane-type valves.     

提高实验猴繁殖率的综合措施

实验猴繁殖率的高低直接影响实验猴养殖企业的经济效益。本文借鉴本中心多年实验猴繁育经验,总结了一套较为完备的提高实验猴繁殖率的措施,内容涵盖实验猴繁殖特性、种公猴选种、繁殖猴分群及梯度饲养、孕猴用药、母猴产后护理、仔猴补饲及断奶、繁殖猴饲料搭配等各方面。这些措施应用于实践后,实验猴繁殖率可保持在较高水平并逐年提高。我们认为这些措施可在其它实验猴养殖企业推广应用。     

Fine needle aspiration cytodiagnosis of subcutaneous sacrococcygeal myxopapillary ependymoma. A case report.

A tumor located subcutaneously over the coccyx of a 32-year-old woman was diagnosed by fine needle aspiration (FNA) cytology as a myxopapillary ependymoma originating in the soft tissue. The FNA smears showed characteristic papillary structures, consisting of a central mucinous core surrounded by cuboidal-to-columnar cells, and scattered ependymal cells. The excised tumor was connected to neither the filum terminale nor the cauda equina. Histopathologic study confirmed the cytologic findings.     

Alveoli increase in number but not size from birth to adulthood in rhesus monkeys

Postnatal developmental stages of lung parenchyma in rhesus monkeys is about one-third that of humans. Alveoli in humans are reported to be formed up to 8 yr of age. We used design-based stereological methods to estimate the number of alveoli (N(alv)) in male and female rhesus monkeys over the first 7 yr of life. Twenty-six rhesus monkeys (13 males ranging in age from 4 to 1,920 days and lung volumes from 41.7 to 602 cm(3), 13 females ranging in age from 22 to 2,675 days and lung volumes from 43.5 to 380 cm(3)) were necropsied and lungs fixed, isotropically oriented, fractionated, sampled, embedded, and sectioned for alveolar counting. Parenchymal, alveolar, alveolar duct core air, and interalveolar septal tissue volumes increased rapidly during the first 2 yr with slowed growth from 2 to 7 yr. The rate of change was greater in males than females. N(alv) also showed consistent growth throughout the study, with increases in N(alv) best predicted by increases in lung volume. However, mean alveolar volume showed little relationship with age, lung volume, or body weight but was larger in females and showed a greater size distribution than in males. Alveoli increase in number but not volume throughout postnatal development in rhesus monkeys.     

Wide range of viral load in healthy african green monkeys naturally infected with simian immunodeficiency virus

The distribution and levels of simian immunodeficiency virus (SIV) in tissues and plasma were assessed in naturally infected African green monkeys (AGM) of the vervet subspecies (Chlorocebus pygerythrus) by limiting-dilution coculture, quantitative PCR for viral DNA and RNA, and in situ hybridization for SIV expression in tissues. A wide range of SIV RNA levels in plasma was observed among these animals (<1,000 to 800,000 copies per ml), and the levels appeared to be stable over long periods of time. The relative numbers of SIV-expressing cells in tissues of two monkeys correlated with the extent of plasma viremia. SIV expression was observed in lymphoid tissues and was not associated with immunopathology. Virus-expressing cells were observed in the lamina propria and lymphoid tissue of the gastrointestinal tract, as well as within alveolar macrophages in the lung tissue of one AGM. The range of plasma viremia in naturally infected AGM was greater than that reported in naturally infected sooty mangabeys. However, the degree of viremia in some AGM was similar to that observed during progression to AIDS in human immunodeficiency virus-infected individuals. Therefore, containment of viremia is an unlikely explanation for the lack of pathogenicity of SIVagm in its natural host species, AGM.     

Transplantation of fetal neocortex in rhesus monkey

A feasibility study of neural transplantation in adult rhesus monkey was undertaken. Fresh and preserved neocortex containing multiplying and maturing neurons obtained from 55–70 gestation days were transplanted into the striatum, cerebellum and cerebral cortex of adult monkeys. Tissues were preserved for 4 days either at subzero temperature in the freezer compartment of the ordinary refrigerator in Ringer lactate or incubated in culture medium. While 2 monkeys out of 5 injected with preserved tissue had successful transplants after 4 months, all the 10 monkeys injected with fresh tissue had no transplants. The size of the two surviving transplants was small. The neurons in the transplants were mainly in clusters. Many of the cells were immature and some showed early degenerative changes. Neuronal processes were restricted to the transplants and thus showed lack of morphological integration with the host tissue. Further studies are in progress to define the nature of the embryonic tissue of primate which can grow and survive and also the role of neural grafts in functional recovery following experimental lesions of the brain regions.     

Influence of needle insertion speed on backflow for convection-enhanced delivery

Fluid flow back along the outer surface of a needle (backflow) can be a significant problem during the direct infusion of drugs into brain tissues for procedures such as convection-enhanced delivery (CED). This study evaluates the effects of needle insertion speed (0.2 and 1.8 mm/s) as well as needle diameter and flow rate on the extent of backflow and local damage to surrounding tissues. Infusion experiments were conducted on a transparent tissue phantom, 0.6% (w/v) agarose hydrogel, to visualize backflow. Needle insertion experiments were also performed to evaluate local damage at the needle tip and to back out the prestress in the surrounding media for speed conditions where localized damage was not excessive. Prestress values were then used in an analytical model of backflow. At the higher insertion speed (1.8 mm/s), local insertion damage was found to be reduced and backflow was decreased. The compressive prestress at the needle-tissue interface was estimated to be approximately constant (0.812 kPa), and backflow distances were similar regardless of needle gauge (22, 26, and 32 gauge). The analytical model underestimated backflow distances at low infusion flow rates and overestimated backflow at higher flow rates. At the lower insertion speed (0.2 mm/s), significant backflow was measured. This corresponded to an observed accumulation of material at the needle tip which produced a gap between the needle and the surrounding media. Local tissue damage was also evaluated in excised rat brain tissues, and insertion tests show similar rate-dependent accumulation of tissue at the needle tip at the lower insertion speed. These results indicate that local tissue damage and backflow may be avoided by using an appropriate insertion speed.     

Risk factors associated with malaria infection identified through reactive case detection in Zanzibar, 2012–2019

Kra monkeys (Macaca fascicularis), a natural host of Plasmodium knowlesi, control parasitaemia caused by this parasite species and escape death without treatment. Knowledge of the disease progression and resilience in kra monkeys will aid the effective use of this species to study mechanisms of resilience to malaria. This longitudinal study aimed to define clinical, physiological and pathological changes in kra monkeys infected with P. knowlesi, which could explain their resilient phenotype. Kra monkeys (n = 15, male, young adults) were infected intravenously with cryopreserved P. knowlesi sporozoites and the resulting parasitaemias were monitored daily. Complete blood counts, reticulocyte counts, blood chemistry and physiological telemetry data (n = 7) were acquired as described prior to infection to establish baseline values and then daily after inoculation for up to 50 days. Bone marrow aspirates, plasma samples, and 22 tissue samples were collected at specific time points to evaluate longitudinal clinical, physiological and pathological effects of P. knowlesi infections during acute and chronic infections. As expected, the kra monkeys controlled acute infections and remained with low-level, persistent parasitaemias without anti-malarial intervention. Unexpectedly, early in the infection, fevers developed, which ultimately returned to baseline, as well as mild to moderate thrombocytopenia, and moderate to severe anaemia. Mathematical modelling and the reticulocyte production index indicated that the anaemia was largely due to the removal of uninfected erythrocytes and not impaired production of erythrocytes. Mild tissue damage was observed, and tissue parasite load was associated with tissue damage even though parasite accumulation in the tissues was generally low. Kra monkeys experimentally infected with P. knowlesi sporozoites presented with multiple clinical signs of malaria that varied in severity among individuals. Overall, the animals shared common mechanisms of resilience characterized by controlling parasitaemia 3–5 days after patency, and controlling fever, coupled with physiological and bone marrow responses to compensate for anaemia. Together, these responses likely minimized tissue damage while supporting the establishment of chronic infections, which may be important for transmission in natural endemic settings. These results provide new foundational insights into malaria pathogenesis and resilience in kra monkeys, which may improve understanding of human infections.     

Spontaneous autoradiographic grain activation associated with mast cells in methacrylate plastic embedded tissue sections

Autoradiographic tracing using tritium labeled compounds or cells is a common laboratory technique for light and electron microscopy. This report describes a chemographic effect associated with certain cells in sections from tissues embedded in the new methacrylate plastic embedding compounds. When tissue sections from rats and rhesus monkeys that received no radioisotope were coated with nuclear track emulsion and subsequently developed, cells with morphologic characteristics of mast cells showed significant grain formation over the entire cell. Three different types of methacrylate plastics were tested using rat and monkey tissues and all three were found to promote grain formation over mast cells; however, this phenomenon was not seen in similar tissue sections from paraffin or epoxy embedded material. The properties of methacrylate plastics which promote positive chemography by mast cells may reflect the greater permeability of this class of plastics. Due to their wide tissue distribution, the presence of such chemographically active cells could cause false estimates of the distribution of either exogenous radiolabeled cells or radioisotopes within many tissues.