**1. Introduction**

Leishmaniasis, a neglected tropical disease, is the third most challenging vector-borne disease after malaria and lymphatic filariasis. According to the World Health Organization's (WHO) published data, it has been reported that leishmaniasis is endemic to approximately 100 countries of the world [1]. Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis. The annual registered cases are around 0.7–1.2 million across the globe. The disease is caused by the protozoan *Leishmania* parasite [2]. The principal vector responsible

**Citation:** Ayari-Riabi, S.; Ben khalaf, N.; Bouhaouala-Zahar, B.; Verrier, B.; Trimaille, T.; Benlasfar, Z.; Chenik, M.;Elayeb, M. Polylactide Nanoparticles as a Biodegradable Vaccine Adjuvant:A Study on Safety, Protective Immunity and Efficacy against Human Leishmaniasis Caused by Leishmania Major. *Molecules* **2022**, *27*,8677. https://doi.org/10.3390/ molecules27248677

 Academic Editors: Marek Brzezi ´nski and Małgorzata Ba´sko

Received: 29 October 2022 Accepted: 29 November 2022 Published: 8 December 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

for mammal-to-human transmission is the Phlebotomine sandfly. Two distinct morphological forms of *Leishmania* have been identified in its life cycle: the promastigote is present in the vector, and the amastigote moves into the monocytes/macrophages of the human or mammalian host [3]. CL is a skin infection that reduces patients' quality of life and imparts psychological problems and social stigmatism [4].

When BALB/c mice, an inbred strain of mice, are infected with the *L. major* parasite, the maturation of a Th2 immune response is triggered by the interleukin-4 (IL-4) produced during the first two days. This IL-4 rapidly renders parasite-specific CD4+ T cell precursors unresponsive to interleukin-12 (IL-12) [5].

Effective regulation requires macrophage activation and nitric oxide (NO) in response to the Th1-produced cytokine IFN-γ. Disease prognosis can be improved by overcoming problems such as low efficacy, systemic toxicity, insufficient drug within macrophages, poor antigen presentation to cells and expensive care. Vaccination may be the most appropriate strategy in this context.

Adjuvants for *Leishmania* vaccines to date are categorized into two types: (i) immunostimulatory molecules and (ii) nanoparticulate and/or delivery systems. The first ones include Bacille Calmette–Guerin (BCG) emulsified with antigens, e.g., *Leishmania amazonenzis* isolate IFLA/BR/1967/pH 8 [6], autoclaved *Leishmania major* ALM or GP63 [7,8], the monophosphoryl lipid (MPL) with LEISH-F1 or LEISH-F2 peptides [9,10], the interleukin-12 (IL-12) with pSP Leish-tec peptide [11], saponin with A2 Leish-tec [12], GLA with SMT Leish-tec [13] and CpG-ODN with KMP-11 Leish-tec [14]. However, immunostimulatory adjuvants suffer from rapid clearance and safety issues [15]. Nano-based delivery systems (NDS) applied as potential adjuvants in anti-leishmanial vaccines may be a useful alternative to conventional bacterial adjuvants and virus vectors [16]. An NDS could potentially deliver target vaccines to the site of action within the host's body, enhance immune reactions by facilitating antigens' absorption and uptake by antigen-presenting cells (APCs) [17], prevent its degradation such as peptides, proteins, or oligonucleotides [18], promote their controlled release and modulate the type of immune responses [19,20]. The particulate adjuvants, for example, liposomes, polymeric microspheres, and emulsions, have been utilized effectively to deliver *Leishmania* antigens in preclinical models of leishmaniasis, as well as other infectious diseases [21]. Liposomes (LPS) and derivatives have been widely studied. Despite the relevance of LPS, some shortcomings are also associated with these lipid-based nanocarriers, such as leakage of the entrapped moiety, oxidation, hydrolysis, and inadequate stability [22]. The polymeric particles, when used as adjuvants, could develop more potent immunogenicity against *Leishmania* antigens [23]. Several polymers, such as polylactide (PLA), polyglycolide (PGA), poly-lactide-co-glycolide (PLGA), poly-caprolactone (PCL), poly-cyanoacrylate (PCA) and natural protein polymers, such as albumin and gelatin, and polysaccharides, have been investigated as vaccine carriers. The polymer that has been explored most extensively is polylactide (PLA). The FDA has approved the use of this compound in human applications due to its biocompatibility and lack of toxicity [23,24]. Currently, PLA is commercialized as part of several nanomedicine tools. For vaccine purpose, antigens formulated with PLA-NPs induce broad and potent humoral and cellular immunity in mice, rabbit and macaque models [25,26]. Other researchers have shown that a mycobacterial antigen adsorbed on lamellar particles of lactide polymers have induced cellular immunity [27].

Another strategy to control leishmaniasis is antigen-target-specific vaccines. These are categorized into three types: (i) live *Leishmania* parasites; (ii) killed *Leishmania* or parasite fractions (first generation), and (iii) *Leishmania* recombinant proteins (second generation) or DNA molecules (third generation) [16]. DNA vaccines are of particular interest because they can effectively induce both CD8+ and CD4+ T cells and produce long-lived antigens and properly folded polypeptides [28]. However, these are still in the early phases of clinical trials. Some trials of developing first-generation vaccines have failed to provide convincing results in phase III due to standardization and safety issues, whereas others are still in earlier phases. Second-generation vaccines, e.g., leish-111f, suffer from a lack

of an appropriate adjuvant [9,21]. Among them, the histone H2B protein was described as a potential candidate [7,10]. The histone protein H2B forms with H2A, H3 and H4, the major constituents of the nucleosome in the nucleus of eukaryotic cells [29]. This protein is conserved among various species of *Leishmania*, i.e., *L. major*, *L. infantum*, *L. donovani* and *L. tropica*. Previous studies investigated the immunogenicity and the protective role of the recombinant H2B protein from *L. major* [30]. H2B, in combination with CpG-ODN, confers effective protection to sensitive BALB/c mice infected with the virulent strain of the *L. major* parasite [31]. Later, Meddeb-Garnaoui et al. showed that this recombinant protein H2B induced a specific Th1-type cellular response in individuals who recovered from cutaneous leishmaniasis infection [32].

The purpose of this research was, on the one hand, to develop a particulate vaccine system based on PLA, a polymer that has received FDA approval, and on the other hand, to protect mice against *L. major* parasite infection. To do this, nanoparticle dispersion was developed using PLA polymer (PLA-NPs). Then, BL21 *Escherichia coli* strain cells were used to express the histone H2B protein, a sub-unit vaccine candidate for leishmaniasis. When animals were experimentally infected with the parasite *L. major* promastigotes, the immune protection of immunized mice was investigated and compared to CpG-ODN, a TLR agonist compound.


**Tadpole:** Not described.

**Distribution** (Figure 169): *Nymphargus mariae* is known from several localities on the Amazonian slopes of the Cordillera Oriental of the Ecuadorian Andes at elevations between 400 and 1078 m ([176,266–268], this work). In Ecuador, the potential distribution of the species is 28,298 km2.

**Figure 169.** Distribution of *Nymphargus mariae* in Ecuador (yellow spots).

**Conservation status:** Globally, *Nymphargus mariae* is listed as *Least Concern* by the IUCN [270]. We agree with this conservation status.

**Evolutionary relationships** (Figure 136): *Nymphargus mariae* is sister to *N*. *colomai* sp. nov.

**Specimens examined:** *Nymphargus mariae:* Ecuador: *Provincia de Pastaza:* 1 km W Puyo (1.4833 S, 78.0 W; 1000–1050 m), MCZ 91187; Andean foothills in the Upper Bobonaza River Basin (ca. 2.0 S, 77.0 W), USNM 291298; stream tributary of Río Lliquino (1.72553 S, 78.98058 W; 400 m), QCAZ 37932; near Villano (1.47445 S, 77.53529 W; 440 m), QCAZ 39293; Sacha Yacu (1.39519◦ S, 77.72946◦ W; 1078 m), MZUTI 183. *Provincia de Napo:* ca. 45 km E of Narupa (ca. 0.729 S, 77.374 W; ca. 800 m), on the Hollín–Loreto road, DFCH-USFQ D285; *Provincia de Orellana:* Reserva Río Bigal (0.52525 S, 77.41785 W; 930 m), QCAZ 48529; Río Huataracu (ca. 0.729 S, 77.374; ca. 800 m), ca. 70 km E of Hollín, on the Hollín-Loreto road, QCAZ 7104, 7499; *Provincia de Sucumbíos:* Lumbaqui (ca. 0.05 N, 77.333 W; ca. 500 m). Peru: *Departamento de Huánuco:* Serranía de Sira (ca. 9.367 S, 74.75 W; 1550 m), KU 174713.

**Localities from the literature:** *Nymphargus mariae:* Ecuador: *Provincia de Pastaza:* Río Pucayacu (1.942 S, 77.042 W) [253]; Conambo (1.86197 S, 76.906 W; 337 m) [176]; 1 km W of Puyo (1.493 S, 78.026 W; 1000–1050 m) [253]; Río Lliquino (1.41486 S, 77.54047 W; 380 m) [176]. *Provincia de Napo:* Río Putuyacu, 45 km E of Narupa (0.734 S, 77.49 W; 800 m) [176,253]. *Provincia de Orellana:* Río Huataraco (1.46986 S, 77.92477 W; 347 m) [176]. *Provincia de Sucumbíos:* Río Verde (0.23786 S, 77.576 W; 726 m) [176]; Lumbaqui (0.04675 S, 77. 34358 W; 515 m) [176].

*Nymphargus manduriacu* Guayasamin, Cisneros-Heredia, Vieira, Kohn, Gavilanes, Lynch, Hamilton, and Maynard, 2019 [21] (Figures 170–173).

*Nymphargus manduriacu* Guayasamin, Cisneros-Heredia, Vieira, Kohn, Gavilanes, Lynch, Hamilton, and Maynard, 2019 [21]. Holotype: ZSFQ 0466, by original designation. Type locality: "Reserva Río Manduriacu (0.310755◦ N, 78.8569◦ W; 1,215 m), Provincia de

Imbabura, República del Ecuador".

**Common names:** English: Manduriacu glassfrog. Spanish: Rana de Cristal de Manduriacu. **Etymology:** The specific epithet "*manduriacu*" refers to the type locality of the species, Río Manduriacu Reserve, Ecuador, a conservation area managed by Fundación EcoMinga (https: //ecomingafoundation.wordpress.com/) [21].

**Identification:** *Nymphargus manduriacu* is easily differentiated from most glassfrogs by lacking webbing between inner fingers (Figure 171) and having, in life, a greyish–green dorsum with numerous yellow spots, which sometimes are surrounded by an ill-defined black ring (i.e., false ocelli; Figure 170). On the Pacific slopes of the Ecuadorian and Colombian Andes, similar species include the following: *N*. *buenaventura*, *N*. *ignotus*, *N*. *spilotus*, and *N*. *luminosus*. *Nymphargus buenaventura* has a light green dorsum with diffuse pale yellow spots; *N*. *ignotus* exhibits a pale tan to olive–brown dorsum with black ocelli surrounding orange or yellow spots; *N*. *luminosus* has a green dorsum with numerous yellow spots; finally, *N*. *spilotus* has an olive green back with small yellow spots [21].

**Figure 170.** *Nymphargus manduriacu* in life from Reserva Río Manduriacu, Imbabura province. (**A**–**C**) Adult male, ZSFQ 0466. (**D**–**F**) Adult female, ZSFQ 0462. Photos by Jose Vieira/Tropical Herping. Obtained from Guayasamin et al. [194].

**Diagnosis:** *Nymphargus manduriacu* exhibits the following combination of traits: (1) Dentigerous process of vomer low or absent, lacking vomerine teeth; (2) snout truncated in dorsal view, and truncated to slight rounded in lateral view; (3) tympanic annulus barely evident, lower three-fourths visible, tympanic membrane colored as dorsal skin, supratympanic fold present; (4) dorsal skin shagreen, with microspicules in adult males; (5) ventral skin granular, subcloacal area with two large subcloacal warts; (6) parietal peritoneum white, iridophores covering one-third to one-half parietal peritoneum (conditions P2 or P3); pericardium white (i.e., covered by iridophores), all other visceral peritonea

clear (condition V1); (7) liver lobed and hepatic peritoneum clear (lacking iridophore layer, condition H0); (8) adult males lacking humeral spines; (9) webbing between Fingers I, II, and III absent, basal between Fingers III and IV (Figure 171); (10) toe webbing basal between Toes I and II, III 11 2 –(21 2 –3−) III (11/3 –11 2 )–(3–3−) IV (3–3−)–(11 2 –2−) V; (11) lacking dermal ornamentations in the form of tubercles, folds, or fringes on hands, arms, feet, or legs; (12) nuptial excrescences Type I and VI; concealed prepollex; (13) Finger I slightly longer than Finger II; (14) diameter of eye larger than width of disc on Finger III; (15) color in life, greyish green to olive green with yellow spots, which, sometimes, are surrounded an ill-defined black ring (i.e., false ocelli); bones green; (16) color in preservative, lavender dorsum with cream spots; (17) iris coloration in life: Light grey with thin grey reticulations and pale yellow hue around pupil; (18) melanophores present and abundant along Fingers III and IV, less dense on Finger II, and rarely present on Finger I; furthermore, present and abundant along Toes IV and V, less dense on Toe III, only at the base of Toes I and II; (19) males call from upper side of leaves; advertisement call is a high-pitched "chirp", with a single, pulsed note with a duration of 0.093–0.118 s ( X = 0.10 ± 0.007; *n* = 10) and a dominant frequency at 4052–4447 Hz ( X = 4267.7 ± 118.3); (20) fighting behavior unknown; (21) egg masses deposited on upper side of leaves, clutch size 15–32 (*n* = 4); no long-term parental care provided by either males or females; (22) tadpoles undescribed; (23) SVL in adult males 24.0–25.7 mm (*n* = 3), and in an adult female 28.8 mm.

**Figure 171.** Hand webbing of *Nymphargus manduriacu* and similar species. ( **A**) *N*. *manduriacu*, ZSFQ 0463, adult male, paratype. (**B**) *N*. *luminosus*, ICN 15930, adult female, holotype. ( **C**) *N*. *spilotus*, ICN 35255, adult female, holotype. Modified from Guayasamin et al. [194].

**Color in life** (Figure 170): Dorsal surfaces greyish green to olive green with yellow spots, with melanophores concentrated around yellow spots, sometimes looking like false ocelli. Upper lip unpigmented. Inner fingers and toes with yellowish hue. Anterior half of ventral parietal peritoneum white, posterior portion translucent. Green bones. Iris light grey with thin, dark grey reticulations and pale-yellow hue around pupil [21].

**Color in ethanol:** Dorsal surfaces of body and limbs grey lavender with small white spots. Parietal peritoneum white, iridophores covering one-third to one-half parietal peritoneum. Heart white (covered by iridophores); all other visceral peritonea unpigmented [21].

**Biology and ecology:** *Nymphargus manduriacu* has only been found at Río Manduriacu Reserve and, although the reserve has been visited several times, *N*. *manduriacu* was only regularly detected during February 2018, with the site experiencing particularly heavy rains on a daily basis [21].

**Call** (Figure 172): The advertising call was described by Guayasamin et al. [21], as follows. Each call is a high-pitched "chirp" that consists of a single note with a duration of 0.093–0.118 s (X = 0.10 ± 0.007; *n* = 10). Notes are clearly pulsed (8–12 pulses per note; X = 10.33 ± 1.366). In each call, there is a slight increase in the dominant frequency with time; the dominant frequency is at 4052–4447 Hz ( X = 4268 ± 118.3). Time between calls is 3.9–8.6 s ( X = 5.72 ± 1.82).

**Figure 172.** Call of *Nymphargus manduriacu*, LBE-C-042, from Reserva Río Manduriacu. (**A**) Oscillogram. (**B**) Audio-spectrogram. Obtained from Guayasamin et al. [21].


*4.1. Developed HCMV VLP Vaccine*

Enveloped virus-like particles (eVLPs) are VLP structures wrapped with a lipid envelope obtained by transfection of the Moloney murine leukemia virus (MLV) gag protein in human or hamster cell lines (i.e., HEK or CHO cells). The MLV gag expression induces burgeoning of particles from membranes of transfected cells. Variation Biotechnologies Incorporated (VBI) laboratories generated an eVLP vaccine expressing an HCMV gB full-length or a chimeric gB protein, where the extracellular domain (ECD) of gB is membrane-anchored using the transmembrane and cytoplasmic domains of the vesicular stomatitis virus (VSV) G protein [90]. The chimeric gB-VSV-G protein is thought to maintain gB in a prefusion conformation. Both vaccines were used to immunize mice and induce a neutralizing antibody response 10-fold higher compared to their soluble recombinant protein counterpart [90]. The vaccine was entered in phase I clinical studies enrolling HCMV-seronegative individuals for evaluation of safety and immunogenicity in early 2016 [91]. An additional eVLP vaccine candidate, targeting both the gB and pp65 antigens, has also been manufactured by VBI. However, we believe that a combination of gB, pentamer, and pp65 or US28 would be more appealing. Finally, another eVLP HCMV vaccine candidate was developed by Redvax GmbH, a Swiss biopharmaceutical company. Unlike VBI, that used mammalian cells, they used the rePAX baculovirus co-expression technology minimizing the purification issues due to the relative large size of VLPs for the efficient generation of a VLP vaccine candidate containing HCMV pp65 and gB [92]. Redvax GmbH was later acquired by Pfizer and the vaccine was able to elicit high-titer neutralizing antibodies and good T cell responses after immunization. Nonetheless, no protection was demonstrated from viremia upon challenge.


Section 2 Humanoid Robots

Mobile Robot, Procedia Manufacturing, Vol. 46, 2020, Pages 509-516, Publisher: Elsevier, https://doi.org/10.1016/j.

*Collaborative and Humanoid Robots*

https://doi.org/10.1177/<PHONE_NUMBER>891329

[21] Chavdarov, I., Krastev, A., Naydenov, B., & Pavlova, G. (2020). Analysis and experiments with a 3D printed walking robot to improve climbing obstacle. International Journal of Advanced Robotic Systems. https:// doi.org/10.1177/1729881420925282

[22] Chavdarov, I., 3D printed coupling between a shaft and a link, patent N 67072 B1, 2020, in Bulgarian, http://ir. bas.bg/fni2018/pdf/patent-67072b.pdf

[23] T. Sun, X. Xiang, W. Su, et al. A transformable wheel-leggedmobile robot: design, analysis and experiment. Robot AutomSyst 98; 2017: 30–41

[14] G. Stano, L. Arleo and G. Percoco, Additive Manufacturing for Soft Robotics: Design and Fabrication of Airtight, Monolithic Bending PneuNets with Embedded Air Connectors, Micromachines 2020, 11(5), 485; h ttps://doi.org/10.3390/mi11050485.

[15] K. Tomczuk, Z. Malecha, 3D printed robotic arm with elements of artificial intelligence, Procedia Computer Science, Vol. 176, 2020, Pages

3741-3750, Publisher: Elsevier, https://d oi.org/10.1016/j.procs.2020.09.013.

[16] M. Tyagi, G. Spinks, and E. Jager, Fully 3D printed soft microactuators forsoft microrobotics, Smart Materials and StructuresSmart Mater. Struct.29 (2020) 085032 (11pp) https://doi.org/

[17] Y. Jia, X. Luo, B. Han, G. Liang, J. Zhao and Y. Zhao, Stability Criterion for Dynamic Gaits of Quadruped Robot, Appl. Sci. 2018, 8(12), 2381; https://doi.

[18] Q. Hao, Z. Wang, J. Wang and G. Chen, Stability-Guaranteed and High Terrain Adaptability Static Gait for Quadruped Robots, Sensors 2020, 20 (17), 4911; https://doi.org/10.3390/

[19] S.Habibian, M. Dadvar, B. Peykari, A. Hosseini, M. Hossein Salehzadeh, and F. Najafi, Design and implementation of a maxi-sized mobile robot (Karo) for rescue missions, Journal reference: Robomech J 8, 1 (2021), Springer, DOI:

10.1186/s40648-020-00188-9.

[20] Chavdarov, I., & Naydenov, B. (2019). Design and kinematics of a 3-D printed walking robot "Big Foot", overcoming obstacles. International Journal of Advanced Robotic Systems.

10.1088/1361-665X/ab9f48.

org/10.3390/app8122381.

s20174911.

**110**

promfg.2020.03.073.

**Chapter 7**

**Abstract**

Kinect

**113**

**1. Introduction**

Humans

Communication and Interaction

between Humanoid Robots and

*Arbnor Pajaziti, Xhevahir Bajrami and Gazmend Pula*

understandably continue to have a firm grip on the main switch.

**Keywords:** Artificial Intelligence, Humanoid Robots, Communication, Interaction,

A variety of humanoid robots have been developed and researched in more than 500 research institutes and universities all over the world. Humanoid robots are robots that have a shape and form resembling that of humans including structural i.e., anatomical similarities such arms and legs as well as facial ones such heads containing eyes and mouths. However the most challenging and complex issue remains the development of the two-legged robots, reliable and capable enough to be meaningful partners to humans, in order to be able to perform actions that humans are capable of, but nevertheless are also needy of having them done all too frequently, especially those of a repetitive kind. Humanoid robots are thus intended to be used repetitive and laborious tasks, frequently more dangerous ones such as permanent inspection, necessary repetitive maintenance ones, especially of a repetitive nature or highly hazardous engagements that may emerge in various types of disasters areas as may be needed in case of emergencies in nuclear power plants.

This paper deals with future robots that will be developed to assist and/or partially replace human activities that would provide for humans very much and frequently needed general-types of repetitive services for their daily tasks and engagements. As indeed the very name of humanoid robots intensely suggests, these engagements despite being routinely self-understood by implication as necessities of daily life, their frequency and repetitiveness, alongside other necessities of distributed elements of an increasingly intelligent daily environment, impose the need for deployment of various kinds of robots. It is to be assumed that there will be middle grounds between different types of humanoid robots, depending on the strength of their field of application. Collaborative robots that are conceived and intended to work i.e., collaborate safely with humans in a joint and shared workspace will expand and develop and be applied in increasingly diverse functions and working environments. Nowadays, intelligent robots are of course widely feasible and also increasingly available, but needless to say, even in the long run they will and cannot surpass the people in their creativity, their ability to learn in their differentiation, and maybe not even manage to catch up with all human complex requirements and needs. People will


**1. Introduction**

It is now more than 60 years since C.P. Snow's [1] *Two Cultures* pointed out that scholars in the humanities and those in the sciences lived in two different worlds. They rarely encountered each other in scholarly contexts and were mostly entirely ignorant about each other's projects. What interested Snow was the scientifically illiterate humanists.

New groups have joined the ranks of the scientifically illiterate, in the eyes of their critics: namely scientists themselves and the educated classes, as well as the policymakers who depend on scientific findings. These newest groups accuse the scientists of ignorance about androcentrism, racism, coloniality, and Eurocentrism that damages the reliability of their results of research. Science is a fully social process, they argue. What we know and do not know is shaped not only by "nature herself," but also by what the most powerful corporations and governments want to know. They point, for example to widespread ignorance about climate change, which has held back effective public policy in this area. And an increasing number of such critics point to the effective non-modern knowledge systems of

non-Western cultures, which have served those cultures well in their distinctive social and natural environments.

Obviously, nobody wants biased research that produces inaccurate accounts of nature and social relations. We want reliable accounts on which to base public policies and our practices. However, this can seem to be a dangerous moment even to take up this question in light of the constant barrage of false claims and "sciencebashing" that issues daily, as I write, from the outgoing U.S. president and other authoritarian regimes around the globe.

Yet today, as the front pages of our newspapers have revealed, who catches COVID-19 and who dies from it indicate that in important respects, maximally objective environmental and medical/health assumptions and practices have not been guiding public policy. COVID-19 is an equal-opportunity virus, but the conditions of life for poor people and peoples of color ensure that they are more likely to catch it and have fewer resources to deal with it. Moreover, in the related economic crisis, who falls into poverty and who does not reveal similar faulty assumptions that shape economic policy.

In response to the earlier complaints, the sciences have corrected their processes in significant ways. As most physicians now recognize, the bodies of members of these other groups are not in all respects exactly like the stereotypical model of the human as the idealized elite white man. Women's bodies are not immature or defective versions of men's bodies, with simply a different reproductive system characterizing them, as the old, pre-1970s accounts claimed.

Engineers also got the message. Automobile designers created the possibility of adjusting the height and position of drivers' seats so that even small drivers, such as many white women and most people in some other ethnic groups, could see out of the front window and at the same time reach the gas or brake pedals. Yet this morning, a story on NPR gave voice to women farmers, who were complaining that tractors and other farming equipment are not user-friendly to anyone but big, very strong men. Manufacturers need to design such items for use by the full array of peoples who farm, including white women as well as men and women in ethnic groups that characteristically are shorter and less heavily muscled.

## **2. Calls for greater objectivity**

Thus, accommodation to servicing the needs of physically and socially diverse groups has produced economic, political, and educational revisions of our policy worlds and our daily experiences in them. These groups want to research that is more objective than the conventional supposedly universally valid research that was grounded only in dominant groups' experiences. They do not want "subjective" research, as their critics often claim. It is the dominant models of the human and their standards that have been only subjective, the critics counter, representing only elite groups' experiences and interests. Rather, they want "stronger objectivity" that can more accurately chart all of our naturally and socially different lives in the worlds that we share.

Some sciences are more liable to such charges than others. High-energy physics certainly seems reasonably resistant to such charges. It does not seem to be at all about people as social beings. Yet one can still ask questions about why it is that these sciences' projects are so highly funded by the U.S Department of Defense. Could this have something to do with U.S. military politics rather than only with the objective desire to understand "pure nature"? Why do not sciences that could effectively prepare for a pandemic—one that on last Friday alone newly infected 99 thousand U.S. citizens and killed 1000—receive equal federal funding? It is becoming clear that today we live in a historically extraordinary moment in which deeply

*Strong Objectivity for New Social Movements DOI: http://dx.doi.org/10.5772/intechopen.99973*

anti-democratic infrastructures have become increasingly visible. Such infrastructures ensure that scientific research will not be maximally objective; it will continue to serve the desires of the powerful at the expense of the needs of the vast majority of the world's citizens. Our standards for objective research that were produced as a result of the earlier social justice movements did not go far enough.


6. Conclusions

This chapter is an attempt to highlight the interferometric techniques used for characterization of optical fibers. Application of two- and multiple-beam interference on different types of fibers is illustrated. Section 2 dealt with conventional optical fibers where we illustrated the theoretical models used to reconstruct the refractive index profiles of these fibers. In these models, the refraction of the light ray traversing the fiber has been considered. Digital holography was explained as an important candidate used for accurate retrieving of phase maps and consequently refractive index profiles of the fibers. In Section 3, we mentioned the problem of fiber bending. Recovering the refractive index profile and mode propagation of a bent fiber considering the refraction of the light rays traversing the fiber is a quite difficult task since bending-induced stresses are responsible for refractive index variations. Also, these stresses are released at the outer surface of the bent fiber. Therefore, we illustrated a successful model that was recently presented to recover the index profile in this case with experimental illustrative data. Another important type of optical fibers is the polarization maintaining optical fibers, which prevent cross-coupling by conserving the state of beam polarization during propagation. In Section 4, we presented interferometric techniques applied on two different polarization maintaining optical fibers, panda and bow tie, to reconstruct their refractive index profiles. Most interference techniques require immersing the fiber in a suitable liquid in order to minimize the phase difference between the fiber and its surrounding medium. In Section 5, an interference technique is presented and applied on a thick optical fiber to recover its refractive index without using an immersion liquid (i.e., in air), which makes the technique suitable for in-situ studying of thick fibers.


**1. Introduction**

Development of restorative therapies for salivary gland dysfunction has been significantly hampered by a lack of accessible and pertinent cell culture models. Salivary glands are made of several cell types, including the saliva-producing acinar cells, ductal cells modifying the saliva as it travels through the lumen, and myoepithelial cells mediating acinus contraction. Acinar cells commonly fail to regenerate following high-dose radiation therapy, aging, or from autoimmune exocrinopathy such as primary Sjögren's syndrome (pSS) [1–3]. pSS is a chronic autoimmune disease characterized by a loss of salivary acinar cell function and a decline in acinar progenitor cell populations exacerbated by lymphocytic glandular infiltration.

Dysfunction of the salivary glands can lead to a reduction in salivary flow, thereby altering saliva composition, pH, and buffering capacity, causing the sensation of dry mouth (xerostomia) [2]. Cell culture models for pSS and xerostomia have been limited, leading researchers to rely on cancer cell lines or short-term cultures of primary salivary gland epithelial cells (SGECs) [4,5]. Moreover, the cell

lines extensively used in salivary research (e.g., HSG, A253, NS-SV-AC) are of male origin [4,6–8]. These cell lines may not recapitulate the pathological processes occurring in pSS, a disease with a higher incidence in female patients [9].

Isolated SGECs have been shown to share ductal and pro-acinar characteristics and exhibit a moderate cell division capacity when grown in serum-free low-calcium media [5,10–13]. As with any primary cell culture model, their limited growth potential can impede reproducibility and applications including, but not limited to, high-throughput drug screening assays. In addition, pSS patient derived SGECs show a markedly reduced proliferation capacity in vitro [3]. The reduced growth capacity of pSS SGECs is mirrored in affected patients' salivary glands where narrowed pools of progenitor cells are found with markedly shortened telomeres [3]. Overall, suboptimal cell culture models combined with the reduced growth ability of pSS SGECs have proven to be major obstacles in salivary research.

Diagnosis of pSS may require a labial salivary gland (LSG) biopsy for histologic analysis of lymphocytic aggregates within the tissue [14]. LSG biopsy tissue is a common explant source for culturing SGECs, but due to limited availability based on clinical access, their potential as a universal model is restricted. Culture of SGECs is typically performed using a dual media approach, where the initial explant medium contains 2.5% fetal bovine serum and is replaced with a second serum-free media to sustain passaging and growth [5,11]. Minor differences in culture conditions, including FBS concentration and time spent in explant medium can have a profound effect on salisphere (i.e., 3D spheroid) formation and passage number, as previously shown for parotid gland progenitor cells isolated from mice [15].

Primary SGEC cultures are comprised of cells with pro-acinar and/or ductal cell-like features depending on the growth substrate, presence of serum in medium, and the specificity of characterization markers used [5,10–13]. The previous work of Fujita-Yoshigaki et al. demonstrated the dedifferentiation of primary parotid acinar cells occurs in culture, which could explain the variety of ductal and acinar cell features expressed [16,17]. In addition, Jang et al. revealed that primary SGECs (known as phmSG) of ductal origin exhibited trans-epithelial resistance, expressed several acinar and epithelial cell markers (i.e., AQP5, SLC12A2, AMY1A), and secreted α-amylase upon β-adrenergic stimulation [5]. Overall, SGECs represent a suitable cell culture model to investigate salivary gland dysfunction in vitro but lack the unlimited growth potential needed for widespread use.

To our knowledge no reliable immortalized human primary SGEC lines derived from female patient's LSG biopsies have been developed or made readily available to researchers. To address this gap, the objective of our study was to generate an in vitro cell culture model for the investigation of the pathophysiology of salivary gland disorders. Using an SV40 Large-T (SV40Lt) lentiviral vector, we generated and characterized immortalized SGECs isolated from LSG biopsies of pSS and xerostomic (sicca) patients. The SV40Lt antigen subunit inhibits both cell cycle regulators, pRB and p53, and prevent telomere shortening-induced cell senescence in some cell types [18,19]. We characterized immortalized SGECs (iSGEC) lines from two non-Sjögren's syndrome (nSS) female patients (referred to as iSGEC-nSS1 and iSGEC-nSS2) and one pSS (referred to as iSGEC-pSS1) female patient, based on the expression of pro-acinar and epithelial cell markers using various molecular methods. iSGECs were grown on matrigel-coated plates to determine three-dimensional (3D) spheroid forming ability, along with the extent to which spheroids recapitulated acinus characteristics, such as differentiated myoepithelial and acinar cells.

## **2. Materials and Methods**


**Author details**

Victor Moskvin\*, Nina Moskvina, Vladimir Nurgaleev, Zhanna Lukyanchikova, Vladimir Nepopalov, Elena Romanina and Vladimir Sopov Department of Psychology, Russian State University of Physical Culture, Sport, Youth and Tourism, Moscow, Russia

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**69**

*Interhemispheric Asymmetries and Individual Features of Regulatory Functions in Sport…*

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[11] Moskvin VA, Moskvina NV. Sports psychophysiology as a new direction of psychology. Sport Psychologist. 2015;**4**(39):10-14 (in Russian)

Hemispheric asymmetry and principles of differential neuropedagogy in higher education. Materials of the International Scientific-Practical Conference for the Workers of Science and Education «Education & Science – 2016». Part 4 St. Louis, Missouri, USA: Science and Innovation Center Publishing House, 2016. p. 259-262. DOI:

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[13] Moskvin VA, Moskvina NV. Psychophysiology and features volitional regulation sport university students. European Journal of Education and Applied Psychology. 2015;**3**:62-65

[14] Moskvina NV, Moskvin VA. Hemispheric asymmetry and features communication processes in sport psychology. European Journal of Education and Applied Psychology.

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[3] Moskvin VA, Moskvina NV. The problem of strong-willed regulation of athletes in the course of preparation of sports psychologists. Materials of the International Scientific-Practical Conference for the Workers of Science and Education «Education and Science-2016». Part 3. St. Louis, Missouri, USA: Science and Innovation Center Publishing House, 2016. p . 9-12.

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[4] Sopov VF. Theory and Methods of Psychological Preparation in Modern Sports. Moscow: Department of Physical Education and Sport Moscow

[5] Puni AC. Psychological Basis of Volitional Training in Sports. Leningrad:

Moscow: FiS; 1962. (in Russian)

[7] Luria AR. Higher Human Cortical Functions and their Disorders in Local Brain Lesions. 3rd ed. Moscow: Academic Project; 2000. 512 p. (in Russian) ISBN 5-8291-0079-7

[8] Luria AR. Basics of Neuropsychology. 5rd ed. Moscow: Academy; 2006. (in

[9] Sakano N. Latent Left-Handedness. Its Relation to Hemispheric and

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HLA-B57 screening test not done at inclusion

Degree of significance between treatment arms \* p< 0,05 \*\* p<0,005 \*\*\*p<0.001

#### **2.3 Resistance development during treatment with anti-retroviral backbones**

Inadequate compliance is the most important cause of virologic failure and resistance development **(23,24)**. Repeated virologic failure with cumulation of resistances will lead to a decrease of the number of CD4 cells (immunologic failure), leading to HIV-related diseases, AIDS and death (clinical failure).

Development of resistance varies per class of anti-retroviral drugs and also within a class of antiretrovirals. For NRTIs, the development of resistance after initiation of cART is stongly associated with adherence. Resistance to antiretrovirals from the start of cART will develop first and during follow-up in highest frequency to lamivudine, followed by development of resistance to NNRTIs, NRTIs and PIs **(32).**

A distinction between the different mutations to NRTI's is made between thymidine analoge mutations (TAMs), TAMs-associated mutations, evolving mainly during virologic failure when on therapy with thymidine analogues ZVD and d4T and the discriminatory mutations and the Q151M pathway mutations conferring for multiresistance. Mutations, an acumulation of mutations or the occurrence of multi-resistant mutations limit the number of effective combinations of NRTIs in the backbone for a second or third regimen.

For instance the K65R mutation (discriminatory mutation) can develop during failing therapy with TDF and will make ABC and ddI ineffective.

These drug-resistant viruses can be tranmitted and may limit the treatment options in treatment naïve patients.

The prevalence of transmitted drug resistance viruses since 2004 is similar in different Eropean countries and the Netherlands. In the Netherlands the prevalence of NRTI drug resistance in recent infection (naïve patients) is found to be around 5-6% and for intermediate or high level of resistance is around 2% **(33)**

Since2003 treatment guidelines recommend onbtaining a genotypic sequence at the start of cART.

#### **3. Safety of anti-retroviral backbones**

366 Recent Translational Research in HIV/AIDS

and less toxic only in the 144-week follow-up data (two trials, 1,119 patients). ABC/3TC had similar efficacy to its comparators, but more AIDS-defining events **(29**). In the Swiss cohort study, including 1318 naïve patients, between 2005 and 2008 drug toxicity remained a frequent reason for treatment modification. Initial treatment with ZVD/3TC was associated with a high rate of drug toxicity. **(30)** In general QoL is beter with a higher CD4 cell count and QoL is decreased in patients with a high VL. The effects of adverse events on QoL are

Third drug ITT analysis:

Table 1. Major characteristics and parameters of effectiviness of the seven important clinical

Inadequate compliance is the most important cause of virologic failure and resistance development **(23,24)**. Repeated virologic failure with cumulation of resistances will lead to a decrease of the number of CD4 cells (immunologic failure), leading to HIV-related diseases,

Development of resistance varies per class of anti-retroviral drugs and also within a class of antiretrovirals. For NRTIs, the development of resistance after initiation of cART is stongly associated with adherence. Resistance to antiretrovirals from the start of cART will develop

trials comparing different backbones of during 48 to 96 weeks of treatment

Degree of significance between treatment arms \* p< 0,05 \*\* p<0,005 \*\*\*p<0.001

**2.3 Resistance development during treatment with anti-retroviral backbones**

atazanavir/r or efavirenz

% patients with VL< 50 cop/mL

% patients with screening

lopinavir/r 68% vs 67% 63% vs 65% 214 vs 193 up to week 96:

efavirenz 67% vs 63% 67% vs 63% 158 vs 163 up to week 48:

efavirenz 78% vs 59%\*\*\* 67% vs 50%\*\*\* 168 vs 134 up to week 60:

efavirenz 82% vs 81% not available 169 vs 167 up to week 48:

similar 75% vs 80% 194 vs 199 up to week 112:

158\*

155\*\*

CD4 increase cells/mm3  % patients stopping study drugs due to adverse events

6% vs 6%

5% vs 4%

up to week 48: 5% vs 19%\* up to week 144: 13% vs 34%\*

up to week 48: 14% vs 18%

14% vs 26%

7% vs 15%

9% vs 9% up to week 96: 14% vs 15%

VL >100.000 copies/mL with VL< 50 copies/mL

efavirenz 77% vs 68%\*\* not available 190 vs

efavirenz 70% vs 69% 67% vs 67% 209 vs

independent of CD4 cell count and VL **(31).**

Treatment arms Number of patients in each treatment arm ()

ABC/3TC (343) versus TDF/FTC (345)

ABC/3TC (388) versus TDF/FTC (393)

TDF/FTC(258) versus ZVD/3TC (259)

ABC/3TC (327) versus ZVD/3TC (327)

ddI/3TC (189) versus ZVD/3TC (187)

ddI/FTC (286) versus d4T/ddI (285)

TDF/FTC (299) versus d4T/3TC (303)


**3. Advantages and limitations of phytoremediation**

Phytoremediation offers several advantages, but also some disadvantages, which should be considered when seeking to apply this technology (Table 1). If low cost is an advantage, the time necessary to observe the results can be long. The pollutant concentration and the presence of other toxins should be within the tolerance limits of the plant to be used. Selecting plants with the efficiency for remediating varied contaminants simultaneously is not easy. These limitations and the possibility of these plants entering in the food chains, should be taken into account when applying this technology.

distribution of nutrients, however species of the genus *Pseudomonas* are the predominant

There are other strategies, which are considered categories of phytoremediation by some authors, but actually, they are mixed techniques or variations of the above mentioned strat‐

**a. Hydraulic barriers:** some large trees, particularly those with deep roots (e.g., *Populus* sp.), remove large quantities of groundwater during transpiration. Contaminants in this water are metabolized by plant enzymes, and vaporized together with water or simply seques‐

**b. Vegetation covers:** Herbs (usually grasses), eventually shrubs or trees, establish on landfills or tailings, are used to minimize the infiltration of rain water, and contain the spread of pollutants. The roots increase soil aeration thus, promoting biodegradation, evaporation and transpiration [7,35-37]. The difficulty of this technique is that tailings generally are not suitable for the development of plant roots. However, various investi‐ gations have been undertaken with the aim of developing processes of cultivation in tailings. For example a technique in which an organic soil composed of sawdust, plant remains, and some NPK-fertilizers is deposited on the surface was utilized by Hungarian agronomists (*Biological Reclamation Process, BRP*), [38]. The workers were able to obtain, at the end of a single biological cycle, 76 different plant species including cereals, shrubs,

**c. Constructed wetlands:** these are ecosystems consisting of organic soils, microorganisms, algae and vascular aquatic plants in areas where the water level is at/near the surface, at least part of the year. All the components work together in the treatment of effluents, through the combined actions of filtration, ion exchange, adsorption and precipitation [27,39,40]. It is the oldest method of wastewater treatment and is not regarded as proper phytoremediation, since it is based on the contributions of the entire system [3,41]. Good cleaning efficiency, low cost of construction along with easy operation and maintenance are the main advantages. It is widely applied in the treatment of domestic, agricultural and industrial waste water, but has proved to be suitable also for treating acid mine

**d. Phytodesalination:** it is a recently reported [13,46] emerging technique that utilizes halophytes to remove excess salts from saline soils. The potential of *Suaeda maritima* and *Sesuvium portulacastrum* in removal and accumulation of NaCl, from highly saline soils, has been demonstrated [47]. Although it has its peculiarities, this technique is a modality

Phytoremediation offers several advantages, but also some disadvantages, which should be considered when seeking to apply this technology (Table 1). If low cost is an advantage, the

organisms associated with roots [13,32,33].

fruit trees and even large trees like oaks and pines.

**3. Advantages and limitations of phytoremediation**

egies. These include:

tered in plant tissues [3,34].

488 Environmental Risk Assessment of Soil Contamination

drainages [42-45].

of phytoextraction.

**Table 1.** Advantages and limitations of phytoremediation [3,7,8,48,49].


**1.1 Hallmarks of systemic lupus erythematosus**

Systemic lupus erythematosus is a well-recognized multi-system disease [1]. Hallmarks of the disorder include the prevalence of antinuclear antibodies (ANA) and double stranded antibodies (DNA). The disease often presents with rashes and/or arthritis or arthralgias. Lupus is "the great imitator," as no organ system is excluded, when diagnosing and treating a lupus patient.


**2.5 Effects of reaction time**

To investigate the effects of reaction time on the morphology of AlN products, the raw materials with 5 wt.% CaF2 were heated at 1800°C and under the N2 pressure of 1 MPa for various reaction times (0.5, 1, 2, and 4 h). **Figure 7** shows the SEM images of the as-synthesized powders.

As the reaction time prolonging from 0.5 to 1 h and further to 2 h, the size and uniformity of AlN particles significantly increased. It was expected that the dissolution and reprecipitation of AlN aiding with liquid phases contributed greatly to the particle growth. In addition, as the prolongation of reaction time, the residual liquid phases in the system could also adjust themselves under the action of the interfacial energy to uniformly wrap the AlN particles, further improving the uniformity of the particle size. However, when the reaction time further increased to 4 h, the growth of AlN particles was no longer obvious. Instead, most of the AlN particles were sintered each other to form large aggregates, and the individual particles tended to change from the spherical to angular morphology, as demonstrated by the

**Figure 7.** *SEM images of the AlN products synthesized for various reaction times: (a) 0.5, (b) 1, (c) 2, and (d) 4 h [29].*

inserted images in **Figure 7c** and **d**. As the reaction proceeding, Ca-aluminates were continuously consumed until they were completely reduced to Ca vapor following Eq. (4). The residual liquid phase was too less to completely encapsulate the AlN particles. The driving force of small AlN particles migrating to large particles decreased, and the energy required for the growth of individual particles increased. Therefore, the AlN particle growth gradually stopped and was replaced by the neck sintering between particles.


**4. Discussion**

The treatment of EP with MTX was established in the late 1980s and has become an alternative to surgical intervention. Recently, cost-effectiveness analysis has put a priority on the patient's subsequent fertility, rather than on detection and/or complication rates, as previously reported (7-9). Effort should be made to confirm the diagnosis of ectopic pregnancy definitively before starting MTX treatment; otherwise, complication rates or costs could rise on the contrary. Seror et al reported that MTX treatment appeared to be costeffective in EP cases where the preservation of fertility was important (10). About 40% of women with ectopic pregnancy are treated medically (11), and 90% of them are successfully treated without any surgical intervention at all (12). Medical treatment can be carried out on an outpatient basis, thus making MTX cheaper than the cost of surgical treatment. Surgical intervention is generally adopted when a high possibility of failure and a prolonged time of resolution is predicted. A particularly high serum hCG level or the presence of fetal heart movement results in medical treatment (13).

It is actually difficult to diagnose interstitial pregnancy correctly because of the slight difference between cornual implantation and interstitial pregnancy. Cornual implantation is usually identified in the upper and lateral uterine cavity, whereas interstitial ectopic pregnancy is within the proximal intramural portion of the tube. Therefore, the accurate diagnosis of an interstitial pregnancy requires precise ultrasound imaging (14). Two regimens are commonly used for the administration of MTX (15) (Table 3). The first contains the administration of MTX and leucovorin on alternate days until beta-hCG concentrations begin to drop. This regimen has a success rate of 93%. The second regimen involves the administration of a single dose of MTX, followed by repeated doses every week if beta-hCG concentrations do not fall by 15% within days 4 and 7. It has been reported that at least 13% of women require two doses, and 1% need more than two; however, more than 90% of women who need a second administration avoid surgical intervention (12). The initial level of hCG is the best prognostic indicator of the need for MTX; however, it is still controversial as to what IU should be the cut-point in deciding whether the administration of MTX is necessary. Lipscomb et al. reported that 94% of 350 women whose initial hCG was less than 10,000 IU/ml had success with MTX treatment, therefore suggesting that an initial hCG level greater than 10,000 IU/ml was a factor in the failure of the treatment (12). On the other hand, Gamzu et al. stated that the cut-point to determine the effectiveness of MTX should be lowered to between 2000 mIU/ml and 3000 mIU/ml (16). The hCG incremental rate both before and after MTX represents an independent risk factor for subsequent tubal rupture. Pre-diagnosis concentrations of hCG which increase at least 66% over 48 hours, followed by persistently rising hCG concentrations after treatment with MTX, may lower the threshold for surgical intervention (17).

It is widely accepted that above the discriminatory zone of 1,500 IU/l-2500 IU/l, a normal intrauterine pregnancy (IUP) should be visible via TVU. At our clinic, we consider an initial hCG greater than 1500 IU/ml and an invisible GS clear indicators of a possible ectopic pregnancy, and an elevation of hCG greater than 1.5 times in 48 hours without GS confirms the diagnosis. Orivieto et al. reported that single-dose MTX treatment for EP does not have a negative effect on ovarian function nor on the outcome of following IVF-ICSI (18). Paul et al. reported that when hCG is >4000 IU/ml, the failure rate of MTX treatment is 65% (17). In this study, the MTX-failure rate of 14 cases where initial hCG was >5000 IU/ml was 7.1% (1/14); however, the failure rate of low hCG cases <5000 IU/ml was 16.6%. In tubal pregnancy, EP become less vascularized as it invades the tubal serosa, and tubal rupture is likely to occur, compared with that in interstitial pregnancy, before MTX treatment can solve the EP. Compared with cases of tubal pregnancy, the difficulty in diagnosing interstitial pregnancy is thought to be the reason why hCG levels are higher at the time of confirming the diagnosis. However, in our study, 14 cases consisted of 7 ampullary, 6 interstitial and one cervical pregnancy, indicating that tubal pregnancy does not always rupture in high-hCG cases. Therefore, more study is necessary to determine the cut-off for MTX treatment.

Recently the cost-effectiveness of treatment has been considered in attempts to save on health insurance costs. Seror reported that, although frequent diagnostic ultrasound is necessary, MTX is cost-effective compared with surgical intervention (10). However, subsequent pregnancies should be followed with extreme caution, and elective cesarean section should be considered as an alternative.

In conclusion, a serum hCG level greater than 5,000 IU/ml is not necessarily resistant to MTX treatment, and interstitial pregnancy is a particularly good candidate for conservative treatment, even if initial hCG is higher than 5,000 IU/ml. However, MTX failure and tubal rupture was identified irrespective of an initial hCG level of 800 IU/ml in tubal pregnancy. Emergent access to surgical intervention must be made available. As well, the patient's awareness of risk and her availability for admission and surgery are equally important. Therefore, preparation for surgical intervention for emergent situations, even after conservative treatment is determined, is essential.

### **5. References**

214 Ectopic Pregnancy – Modern Diagnosis and Management

The treatment of EP with MTX was established in the late 1980s and has become an alternative to surgical intervention. Recently, cost-effectiveness analysis has put a priority on the patient's subsequent fertility, rather than on detection and/or complication rates, as previously reported (7-9). Effort should be made to confirm the diagnosis of ectopic pregnancy definitively before starting MTX treatment; otherwise, complication rates or costs could rise on the contrary. Seror et al reported that MTX treatment appeared to be costeffective in EP cases where the preservation of fertility was important (10). About 40% of women with ectopic pregnancy are treated medically (11), and 90% of them are successfully treated without any surgical intervention at all (12). Medical treatment can be carried out on an outpatient basis, thus making MTX cheaper than the cost of surgical treatment. Surgical intervention is generally adopted when a high possibility of failure and a prolonged time of resolution is predicted. A particularly high serum hCG level or the presence of fetal heart

It is actually difficult to diagnose interstitial pregnancy correctly because of the slight difference between cornual implantation and interstitial pregnancy. Cornual implantation is usually identified in the upper and lateral uterine cavity, whereas interstitial ectopic pregnancy is within the proximal intramural portion of the tube. Therefore, the accurate diagnosis of an interstitial pregnancy requires precise ultrasound imaging (14). Two regimens are commonly used for the administration of MTX (15) (Table 3). The first contains the administration of MTX and leucovorin on alternate days until beta-hCG concentrations begin to drop. This regimen has a success rate of 93%. The second regimen involves the administration of a single dose of MTX, followed by repeated doses every week if beta-hCG concentrations do not fall by 15% within days 4 and 7. It has been reported that at least 13% of women require two doses, and 1% need more than two; however, more than 90% of

Fig. 3. Time-course of serum hCG level in Case II

movement results in medical treatment (13).

**4. Discussion**

[1] Satoe Fujiwara, Yoshiki Yamashita, Sachiko Kawabe, Hideki Kamegai, Yoshito Terai, Masahide Ohmichi. A case of a methotrexate-resistant ectopic pregnancy in which

**13**

*Taiwan*

**The Treatment of Ectopic Pregnancy with**

**Laparoscopy-Assisted Local Injection of**

*1Department of Obstetrics and Gynecology, Taipei Medical University*

*2Department of Obstetrics and Gynecology, Taipei Veterans General*

*3Graduate Institute of Medical Sciences, Taipei Medical University, Taipei*

Ching-Hui Chen1, Peng-Hui Wang2, Li-Hsuan Chiu3 and Wei-Min Liu1\*

Laparoscopy-assisted local injection of chemotherapeutic agents is not yet considered as a standard treatment for ectopic pregnancy. Herein, we demonstrated cases of cesarean scar pregnancy and ovarian ectopic pregnancy successfully treated with trans-vaginal sonography-guided local injection of etoposide. Furthermore, we evaluated the efficacy of laparoscopic local injection of etoposide compared with methotrexate on tubal pregnancy treatment. With the aid of laparoscopic injection, local etoposide treatment offers a precise localization and minimally invasive approach to the management of ectopic pregnancies. Compared to the conventional way to treat ectopic pregnancy using methotrexate, local injection of etoposide is considered to be a high-success rate, low-risk, and less-limitation

option for such types of ectopic pregnancies with careful selection of cases.

ectopic pregnancy (Lurie, 1992; Te Linde, Rock, & Thompson, 1997).

**2. Current opinion and therapeutic strategy on ectopic pregnancies**

Ectopic pregnancy is a complication of pregnancy in which the fetus implants outside the endometrial cavity, which ultimately ends in death of the fetus. It constitutes 1.6% of all pregnancies (Lurie, 1992). Most ectopic pregnancies are located in the fallopian tube, but the implantation can also occur in the cervix, ovaries, abdomen, and even cesarean scars. Ectopic pregnancy is usually not viable. If left untreated, half of ectopic pregnancies will resolve without treatment and presents as the tubal abortions. Ectopic pregnancies are considered as a dangerous health problem for women of childbearing age because of the internal bleeding as a common complication. In fact, maternal mortality is 0.14% in cases of

Within various forms of ectopic pregnancies, cesarean scar pregnancy is a rare form of ectopic implantation. The pathogenesis is thought to be a normal fertilization followed by implantation at the ecchymotic lesion site which is bulging from the uterine wall at the

**1. Introduction**

 \*

Corresponding Author

**Chemotherapeutic Agents**

*Hospital and Taipei Medical University, Taipei*

*Hospital and National Yang-Ming University, Taipei*

dactinomycin was effective as a second-line chemotherapy. Fertil Steril 2009;91:929. e13-15.