Probiotics Use in Horse Feeding - Juniper Publishers

 JOJ Sciences - Juniper Publishers

Abstract

Probiotics are live microorganisms, offered in animal feed at adjusted doses, providing improvements in host health. Several studies have confirmed the yeasts effect on the health, digestibility coefficient, and performance of horses, that way, the Saccharomyces cerevisiae yeast can become an important probiotic additive for equines besides reducing the incidence of colic in these animals.

Keywords: Equine; Saccharomyces cerevisiae; Yeasts; Intestinal microorganisms; Supplementation; Additives normal; Microbiota; Intestinal environment; Digestive tract; Horse feeding; Microorganism; Food digestibility; Gastric environment; Antimicrobial properties; pH; Temperature; Probiotics

Introduction

In equidae, probiotics are used trying to establish a desirable balance among intestinal microorganisms, especially that beneficial. For these benefits, supplementation of these additives in the diet creates an expectation, after use, to provide viable and suitable microscopic organisms for adaptation in the intestinal environment, acting in a beneficial way in the balance of the normal microbiota of the digestive tract, increasing zootechnical performance and/or preventing pathologies of the digestive tract [1]. Therefore, it is essential to evaluate in a more specific way the use of yeasts in the horse feeding as potential probiotics for the specie, considering there are indications of these acting positively on the food digestibility and health of the horses.

Probiotics Characterization

For a microorganism to be considered a good probiotic must have some attributes such as being able to survive the gastric environment, antimicrobial properties, higher growth rate than its elimination by intestinal peristalsis, able to adhere to mucus and epithelial cells, and it is essential to be produced and remains viable for long periods. The first three mechanisms are usually attributed to lactic acid producing bacteria, while the latter two are more yeast specific [1].

The characteristics attributed to probiotic microorganisms are related to the production of substances that suppress pathogens or prevent their growth, competition with them for adhesion sites and nutrients, preventing they to produce toxins as well as inhibiting their action; to be innocuous to the host to not cause any disease, and it must be natural to the digestive system, being able to resist to this environment, mainly to changes in pH and temperature [2].

Several microorganisms are currently used as probiotics, the most common of which are Lactobacillus, Bifidobacterium, Streptococcus, and live yeasts such as Saccharomyces cerevisiae [3]. Saccharomyces cerevisiae yeast has good results, promoting intestinal microbiota balance, which can be observed in an experiment where it was used a dose of 2g/day of yeast for 4-year-old mini-horses, showing balance in the intestinal microflora, and with changes in the bacterial population [4].

Effect of the Saccharomyces cerevisiae Use on the Diet of Horses

Probiotics are widely used for equines in the recovery of animals undergoing some type of stress, such as deficiency in colostrum consumption, stress generated by the weaning process, during transportation with significant water and food restrictions, abrupt climatic changes, chronic morbidities due to mismanagement such as laminitis or gastrointestinal diseases, supplements for animals with low body score, and long-term therapy in order to circumvent the use of antibiotics [5]. In addition, it is used to try to establish a desirable balance between intestinal microorganisms, especially those beneficial. These additives have become a differentiated option due to their attributes, as their ability to reduce infectious pathologies and, consequently, reduce the use of antibiotics [6], as well as to reduce cases of intestinal obstruction. They may be offered to animals in a variety of ways, such as powders, pastes, gels and most often orally, mixed with food during the meal or supplied with water [7].

Among these probiotics, Saccharomyces cerevisiae has potential to be used as a probiotic in equines, since research in other species of animals has shown positive effects [8,9]. In weanling foals, the use of Saccharomyces cerevisiae yeast at 5 g/day showed positive results in hemicellulose digestibility [1]. In diets with low-quality bulky food supplemented with this yeast, the use was approximately 9% better, with an increase in the digestibility coefficient values when compared to the yeast-free diet FURTADO [10]. Similar results were observed when supplementing 20 g of the probiotic Saccharomyces cerevisiae per day for horses under training, with an increase in hemicellulose digestibility of 4.1% [11]. Thus, in order to improve the digestibility of the fibrous portion and increase the energy available to the animals, the diet can be increased with these probiotic additives

Conclusion

The information presented demonstrates that the use of the probiotic Saccharomyces cerevisiae improves the actions of the intestinal microbiota, allowing the greater degradation of the fibrous components of the diet, which predisposes better health conditions to the supplemented animals.

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Tyrosine on Cutaneous Pigmentation - A New Therapy Line for Vitiligo - Juniper Publishers

 Dermatology & Cosmetics - Juniper Publishers

Abstract

The topical effect of tyrosine in altering skin pigmentogenesis was studied using melanin pigment producing strain of Cryptococcus neoformans. Although the biochemical pathway is different in C. neoformans, where phenol oxidase enzyme is involved unlike tyrosinase as in the case of mammalian cell, still the findings prove the method to be reliable and reproducible. Peri-cellular absorption of tyrosine and subsequent melanogenesis were observed in treated cell of C neoformans. Further the quality of pigment produced in the cell of the organism was different from phenol oxidase induced pigment. The pigment thus offered protection to the cells from UV damage. The observation based study on tyrosine based cream in human skin and subsequent sun exposure resulted in minimal erythema and delayed pigment darkening indicating the underlying melanogenesis process. Further, the absence of erythema suggests the possible melanogenesis activation at sub-dermal level and its effect in protecting the skin from sun damage. The clinical usefulness of topical tyrosine and other scientific details are discussed in the paper.

Keywords: Vitiligo; Tyrosine; Phenoloxidase; L-Dopa; Guizotia Abyssinica; Uv Treatment; Pigmentogenesis

Introduction

A rapid, simple, less time consuming, cost effective and finally a reliable and relatable method for screening agents/ingredients and developing a formulation for the management of vitiligo and hypopigmentary disorders of the skin is greatly lacking [1]. Although cell culture methods are available and are used widely for the above purpose, but still the extrapolation of the cell culture findings to predict the possible treatment outcome based on the cell culture assay of agents/ingredients is quite tricky, confusing and in most cases, not relatable [2]. Several microorganisms are known to produce melanin like substance for their defense and the fungi -Cryptococcus neoformans is the best example for melanin synthesis [3]. In general, the microbes synthesize melanin mostly via phenoloxidase but in mammalian system, tyrosinase replaces the role of phenoloxidase. In any case L-DOPA and tyrosine are the precursor molecules for the biosynthesis of melanin [4]. The extracellular supplementation of tyrosine into the growth medium of C.neoformans was found to accelerate the biochemical process of melanogenesis. Further the melanin thus formed was found to accumulate in the cells but a distinct capsule formation around the cell was absent. C. neoformans being a ‘simple life unit’; any changes in the cells of C.neoformans therefore can be easily co-related to the mammalian system [5].

In the case of cell culture assay, where the melanocytes are used but the melanocytes always exist only as an individual cell unit and may not reflect the possible response of the skin in to [6]. Therefore the study outcome that is obtained with cryptococcal cells is more relatable to the cutaneous response than the use of melanocytes. In our present study we have established the extracellular tyrosine induced melanogenesis in cryptococcal cells and such cells are relatively protected from UV induced cell death/mutation. The interesting feature was that the cells grown in tyrosine supplemented media showed greater resistance to UV treatment than the cells grown in guizotia abyssinica agar supplemented with creatinine. Vitiligo being an autoimmune disorder with progression tendency, palliative, tyrosine based therapy may be effective for long term purposes even if the treatment outcome is relatively sluggish [7,8]. In the case of progressive vitiligo, tyrosine based treatment even if it retards the progression of hypopigmentation by 50% still such treatment would be highly valuable. In the present study we report the use of extracellular supplementation of tyrosine and its usefulness in the treatment of vitiligo. Details are presented in the paper.

Materials and Methods

Growth of C.neoformans in tyrosine supplemented media

Sabouraud’s dextrose agar supplemented with 3 different concentrations of tyrosine such as 0.1, 0.2, 0.3 μg/ml respectively were used for the experiment. The freshly grown C.neoformans were inoculated and incubated in the above media condition for 7 days and then observed the colony and the cells for melanin, both macroscopically and microscopically [9,10].

Growth of C.neoformans in guizotia abyssinica agar with creatinine

Cryptococcal cells were grown in guizotia abyssinica agar supplemented with creatinine and incubated for 7 days. After 7 days the colony characteristics and cells were observed for melanin as described above [11,12].

Response of cells to UV

C.neoformans grown in tyrosine supplemented media and from guizotia abyssinica agar supplemented with creatinine were grown again in sabouraud’s agar and were then immediately were exposed to UV for 5 minutes. The number of colonies grown was counted and the melanin synthesized in the cells vis-à-vis UV protection was ascertained.

Melanin quantification

Melanin produced in C.neoformans cells grown in tyrosine supplemented media and guizotia abyssinica agar supplemented with creatinine was quantified using a standard procedure. Approximately 109 cells grown in different experimental setup were pelleted by centrifugation at 1,000 g for 5 min and then the cells were washed with phosphate-buffered saline. After recentrifugation as described above, the supernatant was decanted, the cells were precipitated again, re-suspended in distilled water and 1 ml of ethanol – ether 1:1 (vol/vol) was added to remove the artifact other than melanin. Cell wall lysis procedure was also followed. The insoluble melanin in ethanol–ether was resuspended and centrifuged at 3,000 g for 5 min, the precipitate was dissolved in 1 ml of 1 N NaOH/10% dimethyl sulfoxide at 8 0C for 30 min. The absorbance was measured at 470 nm, and the melanin synthesized in treated and control cells were calculated.

Effect of topical tyrosine on melanogenesis and erythema formation in human volunteer

Five human volunteer were chosen for the study. The cream with tyrosine (0.2%) and without tyrosine was applied over the volar forearm region at 2mg/cm2 separately. Then the volar forearm region was exposed to sunlight for 15minutes and the extent of erythema formed in the treated and control sites and between products with and without tyrosine was recorded immediately after exposure and the pigment production (tanning) in the above experimental set up also recorded after 24 hours [13,14].

Results

Growth of C.neoformans in tyrosine supplemented media

The cells grown in tyrosine supplemented media showed the melanin synthesis which is directly proportional to the concentration of tyrosine used (Figure 1-4). C.neoformans grown in guizotia abyssinica agar supplemented with creatinine did produce intense pigmentation. Microscopic appearance of C.neoformans cells grown in tyrosine supplemented media showed the accumulation of melanin to be higher in cells grown in tyrosine supplemented media with cell wall distinctively with devoid of capsule formation and cell enlargement.

Response of cells to UV

We have used 500 CFU’s of C.neoformans for UV exposure. UV exposure in the case of the organism grown in SDA showed a significant reduction in the colonies. Only 13/500 could grow after UV exposure. Whereas the UV untreated plate yielded 496/500 CFU’s. In the case of organism grown in tyrosine supplemented media when exposed to UV, 76% CFU’s could withstand the UV treatment with only 7.8% of the cells that were grown were mutant colonies. In the case of organism grown in Guizotia abyssinica agar supplemented with creatinine media when exposed to UV yielded only 24% of cells with 93.3% of colonies were mutant.

Melanin quantification

Tyrosine treatment significantly increased the melanogenesis in a concentration dependent manner. The C.neoformans grown in guizotia abyssinica agar supplemented with creatinine also showed melanin synthesis.

Effect of topical tyrosine on melanogenesis and erythema formation in human volunteer

None of the 5 volunteers treated with tyrosine incorporated cream showed erythema response to sun exposure whereas all of them showed varying degrees of tanning after 24 hours.

None of the 5 volunteers treated with the cream devoid of tyrosine showed any tanning response after 24 hours. However all of them showed varying degrees of erythema within 15 minutes of sun exposure.

Discussion

Our present investigation has brought out a new treatment possibility for vitiligo and hypopigmentary disorders of the skin. Tyrosine is although known as the fundamental unit of skin pigment called melanin but still tyrosine is not the sort after treatment option for vitiligo. It is always assumed that amelanotic condition or the ability of the melanocytes to produce the pigment or due to the enzymatic or the biochemical pathway aberrations as the causes for vitiligo. Substrate availability in excess may sometime offer solution to the problem. However which patient may require such treatment option is difficult to predict because the etiopathology of vitiligo and hypopigmentary disorders of the skin is too complex. Therefore simultaneously adopting several treatment options to address various pathological triggers and correcting each of them is necessary. In the present study, we have explored how the extracellular supplementation of tyrosine would increase pigmentogenesis in C.neoformans. In our earlier study we have already established the use of C.neoformans in the screening and identifying the skin pigment modulators as effective as melanocytes based screening methodology.

The advantage of using C.neoformans is that the C.neoformans is a eukaryotic microorganism and also it represents the life in totality. Whereas, the melanocytes are one of the cells in the skin and it requires several direct and indirect communication network with other cells and the feedback mechanism to function. Therefore the scope of predicting the therapeutic usefulness of any compound or agent tested by C.neoformans will have a higher relatability than melanocyte or tyrosinase enzyme based screening outcome. The C.neoformans cells treated with varying concentration of tyrosine has significantly increased the melanogenesis, melanin accumulation and cell wall conspicuous. Further the pigmentogenesis in C.neoformans had occurred in 7 days as against 14 days in the case of guizotia abyssinica agar supplemented with creatinine. Interestingly the capsule formation was fairly less in C.neoformans cells grown in tyrosine supplemented media than guizotia abyssinica agar supplemented with creatinine. The formation of capsule is linked more with virulence and the melanoid formation is linked with the survival of cells.

The pigmentogenesis with poor capsule formation therefore suggests that the tyrosine may be serving only as substrate for the pigmentation and other than that the tyrosine may not alter the virulence mechanism of organism. The absorption of tyrosine from the media by C.neoformans cells clearly suggests the possibility of dermal absorption of tyrosine because the cell wall architecture of C.neoformans is quite impermeable for the easy entry of agents. Therefore a cream with tyrosine may offer better relief to the problems of hypopigmentary disorders; where the substrate abundance is sufficient to promote pigmentation. The microscopic examination of the cells clearly reiterates that tyrosine induces melanogenesis in a dose dependent manner with not much alteration either in the cell size or in the cell wall armamentarium. The cells grown in different growth conditions such as SDA, tyrosine supplementation and guizotia asyssinica agar were exposed to UV to check how the melanogenesis in the cells offer protection from UV.

The cells grown in SDA could not withstand UV exposure and hence majority of the cells could not survive and only a limited number of mutant colonies alone could survive. In the case of tyrosine treatment about 70% of the cells survived UV exposure clearly suggesting that the melanin produced by the cells may be responsible for the UV protection. The countercheck measures on mutant colonies are also revealed only the above possibility. Whereas the cells grown in guizotia abyssinica agar supplemented with creatinine could not withstand UV exposure despite high melanin synthesis. We presume that the phenol shock in the guizotia abyssinica agar may have made the cells vulnerable to UV further the melanin synthesis in the cells in the above media may not be uniform and hence the survival rate of cells vis-àvis mutant formation were almost equal. The quantification of melanin assay has revealed that C.neoformans grown in tyrosine supplemented media yielded pigment which is slightly less blackish than the C.neoformans cells grown in guizotia abyssinica agar supplemented with creatinine.

C.neoformans produces pigment through a different biochemical pathway than the mammalian cells. Phenol oxidase enzyme in C.neoformans which is an analogue of tyrosinase in a mammalian system results in pigment production in C.neoformans. The reason for the poor survival rate of C.neoformans when exposed to UV despite melanin synthesis may be due to the phenol shock in guizotia abyssinica agar medium. The qualitative aspect of the pigment produced by the cells grown in tyrosine supplemented media was significantly different from that of the cells grown in guizotia abyssinica agar supplemented with creatinine; wherein the melanin was densely colored may be due to the capsule inclusion. We presume that the tyrosine may trigger melanogenesis although through phenoloxidase pathway but may be limiting the events just to pigmentogenesis. Whereas the cells grown in guizotia abyssinica agar supplemented with creatinine may pose ‘phenol threat’ to the cells and hence the production of pigment, capsule formation and other associated changes may be taking place in the cells and hence the resultant product is quite dark and dense but still not protecting the cells from UV exposure. Subsequent to the above experiment, we also tried to answer the question of whether topical tyrosine when results in pigmentogenesis also alter the inflammatory responses.

We have used two sets of creams viz., one with tyrosine and without tyrosine. The solar effect on tyrosine supplemented and the cream devoid of tyrosine was checked by applying the creams over the volar forearm region in volunteers and then expose to sunlight. To our surprise we found that tyrosine supplemented cream did not cause erythema formation in response to sun exposure, however resulted in delayed pigment darkening (tanning) after 24 hours. Tyrosine doesn’t filter down UV A selectively and hence the delayed pigment darkening necessarily cue only melanogenesis due to abundant substrate availability. The sun exposure has triggered the evolutionary instinct of the skin to produce pigment and when the substrate was available the pigmentogenesis has taken place. The process involved in melanin pigmentation might have protected the skin and hence no erythema formation was observed in tyrosine supplemented cream was when used.

The above findings further suggest that tyrosine may not only augur melanogenesis in the skin but also may permeate through skin when applied topically. However the delayed pigment darkening in volar forearm region treated with tyrosine supplemented cream disappeared by 48hrs clearly indicating the melanogenesis event was only responsible for tanning and not the dermal protective response to sun exposure. The cream devoid of tyrosine did not offer sun protection and hence the erythema was obvious within 15 minutes of sun exposure. But no immediate or delayed pigment darkening has occurred. The characterization of melanin by Electron Paramagnetic Resonance spectra (EPR) data not included showed that the pigment produced by C.neoformans cells grown in tyrosine supplemented media was distinctly different from that of guizotia abyssinica agar supplemented with creatinine. Topical tyrosine may offer the much needed therapeutic benefit to vitiligo patients as revealed by our study.

However such positive response is possible only in those patients who may suffer from the scarcity of the substrate of melanin- Tyrosine. Differentiating the different etiological causes of vitiligo and accordingly modifying the treatment may not be possible in the case of vitiligo and hence all treatment options such as increased substrate availability, promotion of tyrosinase activity, increased dendrite number and length in melanocytes and accelerating the melanin transfer mechanism to keratinocytes are required. Immunosuppressive agents also required if the active predation of melanocytes by the self -immune defense mechanism were the cause for the pigment loss. Our study has brought out a ray of hope for the treatment of vitiligo. Considering the tyrosine being the last biochemical molecule (amino acid) with decent bio-absorption through skin tyrosine based treatment option may hold key to vitiligo treatment in near future. The cream partitioning co-efficient is necessary for the rapid and sustained release of tyrosine from the cream and therefore it is not just the use of tyrosine alone may offer the result but the formulation science also plays a significant role.

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Dependence of Chlorophyll Content in Leaves from The Light Regime, Electromagnetic Fields and Plant Species - Juniper Publishers

 Horticulture & Arboriculture - Juniper Publishers  

Introduction

The productivity of photosynthesis of green plants is determined mainly by their genetics and strongly depends on temperature, nutrient medium and light regime Polevoj [25]; Andrianova & Tarchevskij [2]. These factors determine the structure and efficiency of the Photosynthetic Apparatus of the plant (PSA). The key functional elements of PSA are a and b forms of the Chlorophyll (Chl) Senge [28]; Kholmanskiy & Smirnov [16] and water, which plays the role of a dynamic matrix and active metabolite Polevoj [25]; Kholmanskiy & Tilov [13]; Aksenov [1]. Water sensitizes PSA by forming complexes with Mg and chiral centers Chl Kholmanskiy & Smirnov [16] and other molecules in the composition of PSA Senge [28]; Kholmanskiy [14]; Kholmanskiy [15]; Zaytseva & Sitanskaya [37]. The abnormal thermodynamic properties of water optimize the physics of cold stratification of seeds at a temperature of about 4°C Kholmanskiy [15] and minimize the energy of seed germination and photosynthesis in the temperature range 15-25°C Polevoj [25]; Kholmanskiy [15]. The photo- and thermophysics of Chl substantially depend on the Mg ion in the center of porphyrin cycle and the electronic nature of substituents in it Senge [28]; Kholmanskiy & Smirnov [16]. In principle, chlorophyll can play the role of a marker of the total Mg content in a plant and therefore can be considered an adequate characteristic of the nutritional value of a plant product.

The photochemistry and photo physics of PSA are determined primarily by the electronic structure of the ground and excited electronic states of Chl. A significant difference in the biophysics of chlorophyll Chl a and Chl b Tyutereva & Ivanova [35] is caused by the replacement of the CH3 group by the electron-acceptor and proton donor groups of CHO in the 7th position of pheophytin (Figure 1). In Frese [9]; Kholmanskiy & Smirnov [16], the electronic nature of the ground and lower excited states of Chl was attributed to states with charge transfers whose dipoles are oriented along the mutually orthogonal X and Y axes of the Chl molecule. The high dipole moments of the excited states of Chls initiate electron and proton transfers from other PSA components and these reactions can accelerate the kinetics of photochemical reactions in PSA. It was suggested in Kholmanskiy [18] that micro polarization of PSA promotes proton diffusion in leaves and intensifies the extraction of micro and macro cell ions by a plant. Mg and Chl greatly increase the nutritional and medicinal value of cultivated plants, both leafy and fruit-bearing. In Kholmanskiy [17-18] it was established that the efficiency of Mg extraction by plant leaves of cultivated plants depends on their species and responds to changes in the irradiation spectrum. It can be assumed that a similar dependence should be observed for the content of Chl in plant leaves. To deepen understanding of the biophysical relationship between plant productivity and Chl we compared the total content of a+b Chl and Mg in raw samples of leaves of a number of cultivated plants and steppe grasses. We analyzed the dependence of the Chl content in the leaves of cultivated plants on the light regime, and also compared the effects on photosynthesis of Chl a and Chl b in plantain and wheat seedlings of an electromagnetic industrial Novichkova & Podkovkin [23]; Shashurin [27] and an electrostatic vortex field Therapeutic reel Mishina [33].

Results and discussion

The empirical data on the content of chlorophylls a and b in cultivated plants and grasses were taken from books and works published by us and other authors. Table 1 shows the total content of Chl a+b (hereinafter [Chl]) and the ratio a/b for raw samples of plant leaves. The determination of [Chl] in the works was carried out according to the methods described in Lichtenthaler & Buschmann [21]. In Bohn [3] [Chl] was determined using liquid chromatography, and the Mg content in Chl was calculated based on its mass fraction of ~2.7% in a and b Chl. In this work, we compared the concentrations of total Mg and Mg in the composition of Chl (Table 2).

A large scatter of [Chl] values in different works is caused by differences in both their measurement methods and the growing conditions of the same plants. The [Chl] values determined for dry samples were recalculated for the wet state using a drying coefficient (k). It was evaluated by determining the weight of a fresh sample and after drying it at a temperature of no higher than 40°C. For vegetable crops (tomato, cucumber), leaf (lettuce, greens) and steppe grasses their k were: 8.5; 15-20 and 6-7, respectively. At the same time, the proportion of water in raw samples was determined by the ratio (k-1)/k and was equal to: 88%; 93-96% and 85-86%, respectively.

The Table 1 shows [Chl] values for plants growing in natural and greenhouse conditions. In the latter case, the age of the samples (weeks) and the light regimes were varied. The irradiators included various combinations of Blue (Bl), Green (Gr), Red (Rt) Light Emitting Diodes (LED1 and LED2) Kholmanskiy [18] and 400W high pressure sodium (SL) and mercury (ML) lamp. Their emission spectra and the absorption spectrum of Chl are shown in Figure. In some works, the LED composition included a diode emitting in the far-red region of the spectrum (FRt) with a maximum at 730nm. Plant species in Table 1 are divided into three groups: vegetables, cereals, berries (I); lettuce and greens (II); grasses (III). The mean values of [Chl] in the II and III groups are close and 1.6 times less than in the I group. At the same time, [Chl-b] in the I-st III-th is 2 times less than [Chl-a], and in the II-th one 3.3 times. The value of [Chl] in the first and second groups of plants reaches a maximum at 3 weeks of growth and depends on the spectral composition and intensity of irradiation. The Сhl content decreases when the irradiation intensity is exceeded by the norms of the optimal light regime Dalke [7]. Moreover, the Chl photo destruction reaction can contribute to the negative effect Kholmanskiy & Smirnov [16]. The efficiencies of Chl biosynthesis in the leaves of plants of group I are close when the plants are irradiated with LED irradiators and the sun and are higher than when irradiated with SL. This result can be explained by the good overlap of the Rt emission bands of LED1 and LED2 with the first absorption band of Chl a and b (Figure 2). In group II, the dependence of the efficiency of Chl biosynthesis on the irradiation spectrum is less pronounced. Chl biosynthesis is limited by the efficiency of the plant’s extraction of the Mg, which depends on the type of plant and the irradiation spectrum Kholmanskiy [18]. Table 2 shows the total content of Mg, Chl, and Mg in Chl ([Mg-Chl]) in the leaves and fruits of plants of the first and second groups, for samples grown under irradiation ML. From these values, the concentration ratio of total Mg to Mg in the composition of Chl (Mg/[Mg-Chl]) was calculated.

The obtained relations Mg/[Mg-Chl] indicate that the total Mg content in plants significantly exceeds [Chl], and this disproportion in plants of the 1st group is 4-5 times greater than in plants of the 2nd group. This is due to the fact that Mg is included in the active center of the enzyme, providing assimilation of CO2 Polevoj [25] and also participates in the activation of many other complexes Senge [28]; Shkol’nik [29]; Sukovataya [32]. In addition, the genetics of cucumber and tomato, unlike lettuce, includes programs for the stages of flowering and fruiting with their specific biochemistry and bioenergy Shkol’nik [29]; Tikhomirov, Sharupich [34]. A significant contribution to the intensification of photosynthesis and enzymatic reactions in animal and plant organisms is made by the magnetic isotope 25Mg Buchachenko [5] the content of which in natural magnesium is 10%. Apparently, 25Mg in the composition of enzymes plays an important role in the functioning of the phytochrome and cryptochrome PSA systems, activating magnetically sensitive dark reactions of radical ion pairs Evans [8]. Chl charge transfer states in dark reactions relax with the formation of long-lived excitons Frese [9]. Their participation in the work of PSA can, in principle, determine the dependence of the efficiency of photosynthesis on external electromagnetic and electric fields. To verify this assumption, we compared the dependence of the content of pigments - Chl a, Chl b and carotenoids (k) in maple leaves growing at a distance of 0 to 1000m (control) from 110kV power lines Novichkova & Podkovkin [23] and in the leaves of plantain seedlings, grown in an Electromagnetic Field (EMF) with a frequency of 50Gz and different intensities Shashurin [27], as well as wheat seedlings growing in an electrostatic vortex field with a frequency of 300kGz Therapeutic reel Mishina [33]. Recent experiments were carried out by us, using similar methods Kholmanskiy & Smirnov [16]; Kholmanskiy [18]. The results and experimental conditions are shown in Table 3. The errors in measuring the pigment content in all samples were about 10%. From the data of Table 3 it follows that the influence of EMF affects the efficiency of pigment biosynthesis only in a tree. The reason for this may be the elongation of transport communications along which charged mineral elements move from bottom to top, and phytohormones from top to bottom. The electrophysical properties of the sap in the layers of cambium and sapwood of deciduous trees, in contrast to the resin of conifers, contribute to electro tropism Kholmanskiy [12].

Conclusion

Thus, it was found that the content of chlorophyll and magnesium in the leaves of cultivated plants is significantly higher in fruitful species than in leafy and, especially in steppe grasses. The chlorophyll content increases when growing plants in light regime using LED irradiators having a radiation band that overlaps well with the first absorption band of chlorophyll a and b. Differences in the content of chlorophyll and plant productivity are associated with a more complex genetic program of metabolism in higher cultivated plants, including the stages of flowering and fruiting.

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Periodic Limb Movements in Sleep (PLMS) and Restless Leg Syndrome (RLS) - Juniper Publishers

 Orthopedic & Orthoplastic Surgery - Juniper Publishers

Abstract

Repetitive leg movements in sleep and Restless Leg Syndrome as two pathologies which can affect various patient groups like ones with neurologic, orthopedic, rheumatic diseases etc. and should be diagnosed and treated properly. There are some signs and symptoms which are related to these pathologies which can help the health care professionals to diagnose these pathologies properly. Also finding the best treatment strategies for these pathologies is of importance.

Keywords: Periodic Limb Movements in Sleep; Restless Leg Syndrome

Abbreviations: PLMS: Periodic Limb Movements in Sleep; RLS: Restless Leg Syndrome

Introduction

Repetitive leg movements in sleep and Restless Leg Syndrome are two pathologies which can be seen in various patient groups including ones with neurologic, orthopedic, rheumatic diseases etc. This is a brief review on these pathologies [1-2].

Discussion

Repetitive leg movements in sleep which last about 20 to 40 seconds and occur hundreds of time which cause awakenings during the sleep and consequently fragmented sleep, are the characteristics of the Periodic Limb Movements in Sleep or PLMS [3-4]. Insomnia can be seen in the patients with PLMS either. Periodic Limb Movement index, which is defined by the limb movements number in an hour of sleep, can be used for PLMS diagnosis. Leg movement study during the night with ten or more movements during an hour, can be used for clinical diagnosis of the PLMS. PLMS can be seen in the patients with narcolepsy, sleep-disordered breathing and fibromyalgia [5-6]. PLMS prevalence in adults is about 5 to 6 percent, although this rate would increase with age in a dramatic manner. For example, this rate can be increased to 45 percent in the patients with more than 65 years of age.

Leg dysesthesia or a creepy crawling or restless sensation which occur in a relaxed and awaked state and will be relieved by limb movement, is a characteristic of a syndrome which is called Restless Leg Syndrome (RLS) which is strongly related to the PLMS. Restless Leg Syndrome can be diagnosed by taking history from the patient or his/her bed partner by questioning about having restless and unpleasant feelings in their legs during relaxation or sleeping in the evening or at night which would be relieved by limb movement or walking. Since peripheral neuropathy, anemia or uremia can cause or exacerbate the restless leg syndrome, patients with RLS should be evaluated for having these concomitant problems either [7-9]. Dopamine agonists can be used to treat PLMS and RLS. Pramipexole and Ropinirole are the medications which are approved to treat Restless Leg Syndrome.

Conclusion

It is important for the health care professionals to have enough knowledge about PLMS and RLS, to treat the affected patients better during clinical practice.

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A Significant Presence of Pyrogen in Blood Bags was Revealed in a Pilot Study - Juniper Publishers

 Blood Research & Transfusion Journal - Juniper Publishers

Abstract

Pyrogens are substances that are typically produced by bacteria, which, when introduced into the bloodstream, can cause pyrexia (fever). The presence of pyrogens in injectable fluids such as anticoagulant solutions in blood bags and water for injection could cause unexplained fever in patients transfused or injected with such fluids. We designed this study to screen for the presence of pyrogen in anticoagulant solutions in blood bags. Chemical method was used to test for the presence of pyrogen (endotoxin) in anticoagulant solutions of 40 blood bags. 1% potassium permanganate was prepared using the sample to be tested, 500 microliters of the 1% potassium permanganate were mixed with 10ml of the samples to be tested and boiled for 30 minutes. Colour change was used as an indicator for measuring the pyrogenic activity. Data were analyzed using simple percentage calculations. The result revealed a significant presence of pyrogen in blood bags. Fifteen out of the 40 anticoagulant solutions in blood bags, representing 3 out of 8 types of blood bags (37.5%) were found to contain pyrogen. Earlier data and this demonstrate that some of the fevers reported in patients following blood transfusion may be caused by the presence of pyrogen in the blood bags, not necessarily due to serological mismatched. Therefore, a full-scale study in this area will help to provide the basis for adequate quality control measures to be put in place to verify the sterility of blood bags for routine use in blood transfusion.

Keywords: Pyrogen; Anticoagulant solutions; Potassium permanganate; Fever; Therapeutic Benefits; Serological; Cryoprecipitate; Morbidity; Staphylococcus

Introduction

Blood transfusion is an essential routine life-saving medical procedure in which a compatible blood or blood product is given to patients that require such a form of medical care [1,2]. This procedure provides remarkable therapeutic benefits, especially to anemic medical and surgical patients [1,3]. Although blood transfusion is used in various medical conditions to help in replacing whole blood loss or blood products, such as, red blood cells concentrate, platelets concentrate, fresh frozen plasma and cryoprecipitate or plasma derivatives such as albumin, coagulation factors and immunoglobulins, the intervention is not without the potential hazards of acute or delayed complications such as acute hemolytic transfusion reaction and transfusion-transmissible infections respectively [4,5]. Notable signs of reactions following blood transfusion may include fever, chills, pruritus, or urticaria as well as severe shortness of breath, red urine, and even loss of consciousness [6]. More severe and potentially fatal acute or delayed transfusion reactions have been reported [7].

Pyrogen refers to substances, typically of biological origins such as bacteria (basically gram-negative endotoxin), viruses, and fungi, that can cause pyrexia when injected into the body via parenteral routes; for instance, the intravenous (IV) [8-10]. Some characteristics of pyrogens include a very low molecular weight that allows them to pass through a semi-permeable membrane; they are also thermostable and filterable. When sufficient quantity of pyrogen is present in the body, it can lead to severe signs of inflammation, including fever, shock, multiorgan failure, and sometimes even death in humans [8]. The best-studied pyrogen is the lipopolysaccharide found in the membrane of gram-negative bacteria. However, bacteria are not the only sources of pyrogen; they can also be derived from plants and animal tissues [11]. Pyrogenic contamination of solutions for parenteral use, such as in blood transfusion anticoagulant preservatives and injection water has posed serious threats to medical care [8]. The major contaminants which contribute to pyrogenic response and complications used in vivo or in vitro are endotoxins. These endotoxins are not easily inactivated by heat as such heat treatment of toxins contaminated products does not help in combating the threats they pose [12].

Transfusion pyrexia (fever) is one of the important clinical symptoms which may occur as an isolated event or as part of complications associated with blood transfusion [13,14]. Fever has been reported as one of the principal causes of morbidity and maybe an essential sign of life-threatening complications of blood transfusion [13]. Fever is often a reason why blood transfusion may be discontinued abruptly, and unfortunately, adequate evaluation of this has remained a thing of concern to the clinicians and other healthcare providers [13].

It is, speculated that the presence of pyrogen in blood bags could be responsible for some of the transfusion pyrexias and idiopathic fevers that could be life-threatening to the patient. This pilot study, therefore, aimed to screen for the presence of pyrogen in anticoagulant solutions in blood bags sold in some pharmaceutical stores within Jos metropolis. It hoped that the outcome of this study might provide some useful insights that can help in the design of a largescale study that will employ both the chemical and biological methods to screen for the presence of pyrogen in blood bag anticoagulant preservatives. The proposed full-scale study will cover more pharmaceutical stores in Jos metropolis, and wider makes of blood bags will be analyzed.

Materials and Methods

Study location

This study was carried out in Jos metropolis in Jos North Local Council of Plateau State. Jos is the capital city of Plateau State, which is located in the Middle Belt region of Nigeria. The city has a population of about 900,000 residents based on the 2006 census. Plateau state has a size of 26,899 km2, with a population of 3.5million and the twelfth largest state in Nigeria. The State lies at latitude 9°55’N and longitude 8°55’E. The highland rises from 1,200 at the lowlands to a peak of 1,829metres above sea level. It has a near temperate climate with an approximate mean temperature of 22°C and the lowest temperature of 18°C. The mean annual rainfall varies from 131.8 to 146 cm [15,16].

Experimental design

Blood bags were purchased randomly from pharmaceutical stores in Jos metropolis. A pack of 5 Citrate-Phosphate-Dextrose (CPD) Blood bags from different vendors/companies were purchased from pharmaceutical stores code numbered 1-5, in Jos metropolis. We screened the anticoagulant solutions of the five bags each of make coded A, B C, D, E, F, G and H for the presence of pyrogen. A controlled study was carried out prior to the commencement of this study where five positive and five negative samples were boiled at 100°C for at different time intervals of 20, 30, 40, 50 and 60 minutes respectively. A colony was obtained using the McFarland’s standard from an overnight incubation of Staphylococcus aureus and introduced into the five positive control tubes before heating in pair with the negative control for the various time intervals.

Inclusion and exclusion criteria

All the blood bags screened were inspected at the point of purchase to ensure that there were no breakages or damages of any sort. Any blood bag that physical integrity could not be ascertained or expired was excluded from this study.

Study sample size

This is a pilot study, aimed at conducting a small-sized experiment that can help in planning and provide justification for a future large-scale study in this subject area [17]. Therefore a sample size of 40 was considered adequate for this work.

The anticoagulant solutions & materials

Each CPDA (Citrate-Phosphate-Dextrose-Adenine) blood bag contains about 63ml of anticoagulant solution for a final anticoagulant: blood ratio of approximately 1:7 in a 450ml wholeblood unit. We used pyrogen-free glass khan tubes, Nickel-Electro 4litre unstirred water bath with a thermometer and a plastic lid. Other materials used included a mechanical analogue stop clock, 100ml measuring cylinder, automatic pipettes and five-milliliter sterile plain tubes.

The laboratory experiment & principle of the test

Chemical method was adopted to screen for the presence of pyrogen in the anticoagulant solutions using potassium permanganate (KMn04). The principle of the experiment is based on the fact that potassium permanganate is a potent oxidizing agent, which is reduced from its tinge purple colour to colorless in the presence of organic matter such as bacteria endotoxin (pyrogen) and plants by-products, after boiling for 30 minutes. If the colour remains tinge purple after boiling, it indicates a negative result, but when it turns colorless, it is an indication that pyrogen may be present [18].

Preparation Of 0.5% Mcfarland standard

A 1.2% of Barium chloride (BaCl2) was prepared by dissolving 1.2g of BaCl2 salt in 100ml of water. 1% sulphuric acid (H2SO4) was prepared by adding 1ml of sulphuric acid to 100ml of water. From the solutions prepared, 99ml of the diluted sulphuric acid was dispensed into a clean beaker, and 1ml of BaCl2 solution was added to the 99ml of H2SO4, and the optical density was read. This solution was stored and used as a reference standard for picking a colony of Staphylococcus aureus being introduced into the positive control in each batch of the samples tested, as shown in (Figure 1). A 0.5 McFarland standard provides an optical density comparable to the density of a bacterial suspension 1.5×108 colony-forming unit (CFU)/ml [19,20]. In other words, 1.5×108 cells suspension was used in each of the positive controls.

The test procedure

One per cent (1%) potassium permanganate was prepared by dissolving 0.1g of the crystalline KMn04 in 10ml of the anticoagulant to be tested. From the solution of 1% KMNO4 prepared, 500 microliters were dispensed into a sterile test tube, 10ml of the anticoagulant preservative solution to be tested was added to the solution above, and the mixture was boiled for 30 minutes. The boiled mixture was observed for colour change. This same procedure was used to test the remaining blood bags.

Data analysis

The prevalence of pyrogen in the total number of blood bags screened and in the types of blood bags screened was determined using simple percentage calculations.

Results

Eight different types of blood bags were purchased from some Pharmaceutical vendors and stores in Jos metropolis as depicted in (Table 1). A pack of five blood bags was purchased from each Vendor and a total of 40 blood bags were screened for the presence of pyrogen.

As shown in (Table 2) of the eight types of blood bags coded A – H that were collected, 37.5% (representing 3 types of blood bags) tested positive for pyrogen while62.5% tested negative. Even though there was no statistically significant difference between the number of the types of blood bags that tested positive to those that tested negative (ꭓ2 = 0.500; p = 0.480), data showed that close to half of the types of blood bags tested positive. Also, of the 40 blood bags that were collected, 37.5% tested positive for pyrogen 62.5% tested negative as shown in (Table 3). However, there was no significant difference between the total number of blood bags that tested positive for pyrogen and those that were negative statistically (ꭓ2 = 2.500; p = 0.114).

Discussion

This experiment was designed to evaluate for the presence of pyrogen in the anticoagulant solutions in CPDA blood bags. The pilot study was aimed at testing the hypothesis which states that, the presence of pyrogen in anticoagulant solutions in blood bags could contribute to the increased cases of fevers (blood transfusion reactions) following blood transfusion. The data presented in tables 2 & 3 revealed a markedly increased presence of pyrogen in the anticoagulant solutions in blood bags of 37.5%. The study further showed that only 62.5% of blood bags in circulation in Jos metropolis were pyrogen-free. The finding of this study further suggests that the presence of pyrogen in anticoagulant preservatives could contribute significantly to febrile blood transfusion reactions. Kenedy and colleagues in their study, which comprised of 90% of patients who had total knee replacement surgery reported that the risk of developing fever increases four times for each unit of blood transfused [21]. The study by Kennedy [21]. did not point to the factors that pyrogens are associated with increased fever following the transfusion of each unit of blood. From the finding of this pilot study, the presence of pyrogens in the blood bags may be responsible for such increased in fevers reported.

The study by Payne and Rolfs believed that febrile nonnonhemolytic reaction was mainly due to antileucocytes antibodies directed against HLA antigens or granulocytes- specific antigens [22]. Contrarily to the beliefs by Payne and Rolfs, a group of researchers found that there was no significant impact of leucodepletion of red cells on the incidence of febrile nonhemolytic transfusion reaction [23]. Transfusion pyrexia could be a sign or symptoms of the Systemic Inflammatory Response Syndrome (SIRS) complication associated with transfusion episode due to bacterial contamination of blood for transfusion [24]. This study correlates with the finding our work that observed the significantly high presence of pyrogen in blood bags tested; these both underscore the need for increased sterility surveillance during the preparation of blood bags especially the fluid use for the anticoagulant storage and transportation.

Another related study carried out by Kindergarten, which evaluated the air-borne pyrogens based on human whole blood cytokine response, reported that the product identity was pyrogen-free, and there was no statistical difference. Also, in another similar study conducted by Hindman. It was shown that most of the injection water they tested show no statistical significance pyrogen presence (p value>0.05) [25]. This study is not free from limitation as the experiment was only designed as a pilot study in preparation for a large-scale study. Therefore, there is a need to fund further works in this field in order to generate data that could significantly improve the quality of blood transfusion services for better management of patients.

Conclusion and Recommendation

Blood transfusion services are an integral part of medicine, therefore quality control mechanism needs to be put in place to guarantee pyrogen-free blood and blood product transfusion for effective management of patient requiring such services. Every health facility producing and or using blood products should ensure that quality control systems are in place to ascertain the sterility of such blood products before they are being transfused. A full-scale study on the subject, covering regions and increased varieties of blood bags in routine use, will provide an insight into the roles of pyrogens in complications of blood transfusion. As a part of the future study, blood bag makes that are associated with fever in patients during or soon after transfusion will be investigated for pyrogen.

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Cooling of Tree Surface Area due to Solar Reflective Coatings - Juniper Publishers

 Engineering Technology Open Access Journal - Juniper Publishers  

Introduction

The application of a high solar reflectance coating to tree trunk surface area reduces the temperature of the surface when exposed to heat input from the sun. Solar reflectance (r) of a surface is a number between 0 and 1 that quantifies the fraction of incoming solar radiation that is reflected by a surface. The solar absorptance (α) is the fraction of solar radiation that is absorbed by the surface. The solar absorptance is 1 – r. Coatings with solar reflectance’s above 0.8 are often referred to as “cool coatings”. The α value foe cool coating is generally less than 0.2. High solar reflectance, cool, coatings have found use is reducing the maximum day-time temperature of roofs (cool-roof coatings). The same technology has potential use for the reduction of maximum day-time temperature of tree-surface area. A reduction of maximum tree surface temperatures contributes to the long-term health of the tree.

Heat Balance to Calculate Temperatures

The surface temperature of a surface exposed to solar radiation, I , can be calculated from a heat balance as shown in the ASTM Standard Practice E 1980 [1]. The heat balance as shown by the following equation includes the input of heat due to solar radiation (α∙I), thermal radiation exchange between the tree surface and the sky (ε.σ.(T_s⁴−T_SKY⁴)), and heat exchange by conduction and convection between the tree surface and the surrounding air (h_C.(T_S–T_AIR)).

Since radiative transport is involved, the temperatures are expressed on the absolute or Kelvin scale (K).

All of the terms in Equation are specified except T_S, the tree surface temperature on the Kelvin scale. T(°F) is obtained from T(K) by Equation 2.

T_S is the “unknown” in Equation 1. The values for TS that satisfy Equation 1 can be determined numerically by assigning trial values for T_S until the right side of Equation 1 equals zero. The T_S values depend on the numerical values of all of the other terms in the equation. The values selected for the terms in Equation 1 are shown in Table 1. The values selected are guided by the discussion in E 1980. The combination α∙I will be shown as Q_SOL to signify heat input from the sun in W/m2. The wind speeds shown in the h_c line are approximations.

Results for Tree-Surface Temperatures

Table 2 contains surface temperature, TS, that satisfy Equation 1. for the listed TAIR, QSOL, ε and hC values. The smaller hc value (5) represents very little air movement. An increase in air speed to attain hC 17 results in significant cooling of the tree surface (212.5 to 160.9, for example).

Table 3 contains the results of subtracting TAIR from each of the entries in Table 2. The results show that the air speed (indicated by the increase in hc ) has a major impact on the tree surface temperature (cooling effect of the moving air). The horizontal line shows that temperatures for a coating with high r (r=0.9) and high solar flux (1000). The tree surface temperature is close to the air temperature even though the solar flux is very high. The negative values occurring at night (QSOL=0) represent cooling by radiation to the night sky. The temperatures in Table 3 are °F.

Table 4 contains temperature reductions due to changing the solar absorptance (a) from 0.9 to either a=0.1 or a=0.2. These results are based on equations that are derived from the data shown in Table 2 and Figure 1.

The temperature reductions due to coating in Table 4 are in °F. The uncoated surface is assigned a solar absorption of 0.9. Two values for the solar absorption of coated surface were evaluated to show the range anticipated for the temperature reduction due to coating. Figure 1 contains the surface temperatures for the four cases that were considered. The graphical results show the distinct division for the two heat transfer coefficients (h or hC) for which results were calculated. The Surface temperatures are directly proportional to the absorbed solar radiation when the other terms remain constant.

Discussion

The calculations presented here do not include heat conducted to the interior of the tree or the cooling effect of evaporation. Bark temperature of 130°F have been reported for wild cherry trees by Sheppard et al. [2] for air temperatures in the range considered here but unknown solar flux. The 130°F observation corresponds to the current results with solar flux of ~500W/m2 and r = 0.2. The high temperatures obtained in the present calculation result from severe conditions: high solar flux and very little wind.

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Pre- and Post Treatment Computed Tomography Finding of Nasopharyngeal Carcinoma - Juniper Publishers

 Clinical & Medical Imaging - Juniper Publishers  

Abstract

Background: Nasopharyngeal carcinoma, the most common neoplasm to arise in the nasopharynx, is a locally aggressive tumor with a high incidence of cervical nodal metastases. This study aimed to investigate the role of computed tomography in the evaluation of nasopharyngeal carcinoma before and after treatment.

Methods: 338 patients with an initial diagnosis of nasopharyngeal carcinoma before and after neoadjuvant chemotherapy were enrolled. All patients were undergone pre- and post-treatment CT scan.

Results: The 338 patients included in this analysis comprised 92 men and 246 women with a median age at diagnosis of 49 years (range, 10–89 years). WHO histopathologic type of non-kerаtinizing undifferentiаted cаrcinomа was acounted 281/338 (83.1%), non-kerаtinizing differentiаted cаrcinomа was 15.1%, and kerаtinizing squаmous cell cаrcinomа was 1.8%. With CT scan staging, the proportions of T1, T2, T3 and T4 were 8.9% (30/338), 17.7% (60/338), 32.8% (111/338) and 40.5% (137/338), respectively; the proportions of N0, N1, N2 and N3 were 12.7% (43/338), 31.9% (108/338), 45.0% (152/338), and 10.4% (35/338), respectively. After treatment, a persistence of the disease was found in 14% of patients, while a recurrence of the cancer was diagnosed in 5% and distant metastasis developed in 11% of the patients.

Cоnclusiоn: Computed tomography is essential for detection of early NPC, staging of the primary tumor, and follow-up.

Keywords: Computed Tomography, Nasopharyngeal Carcinoma, Radiotherapy, Chemotherapystrictures

Intrоductiоn

Nаsophаryngeаl cаrcinomа (NPC), the most common neoplаsm to аrise in the nаsophаrynx, is а locаlly аggressive tumor with а high incidence of cervicаl nodаl metаstаses. The tumor hаs а propensity towаrds extensive invаsion into аdjаcent tissues, pаrticulаrly lаterаlly into the pаrаphаryngeаl spаce аnd superiorly into the skull bаse. However, spreаd to the pаlаte, nаsаl cаvity, аnd orophаrynx hаve аlso commonly reported. Distаnt metаstаses cаn аrise within bone, lung, the mediаstinum аnd, more rаrely, the liver [1-3]. Аlthough NPC is rаre in North Аmericа аnd Europe with аn incidence of 0.5-2 per 100,000, intermediаte incidence rаtes аre seen in Southeаst Аsiа, the Mediterrаneаn Bаsin, аnd the Аrctic rаnging from 0.5 to 31.5 per 100,000 person-yeаrs in mаles аnd 0.1 to 11.8 person-yeаrs in femаles [4-6]. In southern Chinа, NPC is endemic with overаll NPC incidence rаtes reаching 20-30 per 100,000 person-yeаrs аnd 15-20 per 100,000 person-yeаrs аmongst mаles аnd femаles, respectively, in the province of Guаngdong [7,8]. NPC hаs а mаle to-femаle rаtio of 2-3:1 [9,10], аnd is most common аmong pаtients 40–60 yeаrs old with bimodаl аge peаks in the second аnd sixth decаdes of life [11,12].

Eаrly detection, eаrly diаgnosis, аccurаte stаging аnd evаluаtion аfter treаtment hаd been the key to improve the efficаcy of treаtment аnd prolong survivаl period [13]. CT hаs been the most reliаble аnd well-estаblished imаging technique for stаging аnd аssessing the extent of nаsophаryngeаl cаrcinomа. The biggest аdvаntаge of CT imаging is thаt the surrounding bony destruction by NPC cаn be cleаrly visuаlized on CT imаge [13]. Zheng et аl. [14] found thаt rаdiologists should pаy аttention to bony structures invаded by nаsophаryngeаl cаrcinomа on CT imаges. In аddition, CT scаn is very quick аnd аbundаnt informаtion cаn be аchieved, it is аlso relаtively cheаper аnd therefore is still аn effective imаging method for diаgnosis аnd follow-up of nаsophаryngeаl cаrcinomа [15,16]. This study аims to investigate the role of computed tomography in the evaluation of nasopharyngeal carcinoma before and after treatment.

Mаteriаl аnd Methods

From July 2015 to April 2019, а totаl of 338 consecutive pаtients with NPC visited HCMC Oncology Hospitаl, Vietnаm. Аll pаtients with histologicаlly proven NPC newly diаgnosed were enrolled in this study. They completed а pre- and post-treаtment evаluаtion thаt included physicаl exаminаtion, nаsophаryngeаl fiberoptic endoscopy, CT scаn of the nаsophаrynx аnd neck, chest rаdiogrаphy, аbdominаl sonogrаphy.

The CT scаns were obtаined with а GE Optima CT660, in аxiаl plane аnd coronаl, sagittal recontruction аfter the injection of contrаst medium (Ultrаvist 370, Schering, Berlin, Germаny) using 3 mm section thickness; 50 mL of Ultrаvist wаs аdministered by intrаvenous.Аll the imаges were reviewed аnd аssessed by two of the аuthors independently. Cаses with vаriаble interpretаtion or disаgreement in stаging between the observers were reevаluаted side by side, аnd the differences were confirmed to reаch а finаl consensus. Medicаl records аnd imаging studies were аnаlyzed, аnd аll pаtients were restаged аccording to the 8th edition of the Union for Internаtionаl Cаncer Control/Аmericаn Joint Committee on Cаncer stаging system [17] relies on evаluаtion of the primаry tumor (T cаtegory), the drаining nodаl groups (N cаtegory). Histopаthologic evаluаtion wаs аccording to the 2003 World Heаlth Orgаnizаtion clаssificаtion: non-kerаtinizing undifferentiаted cаrcinomа (NKUC), non-kerаtinizing differentiаted cаrcinomа (NKDC), kerаtinizing squаmous cell cаrcinomа (KSCC).

SPSS version 20.0 (IBM, Аrmonk, NY) wаs used for аll stаtisticаl аnаlyses.

Results

The 338 pаtients included in this аnаlysis comprised 92 men аnd 246 women with а mediаn аge аt diаgnosis of 49 yeаrs (rаnge, 10–89 yeаrs). WHO histopаthologic type of NKUC wаs аcounted 281/338 (83.1%), NKDC wаs 15.1%, KSCC wаs 1.8%. Аccording to the eighth edition UICC/АJCC stаging stаndаrds, the proportions of T1, T2, T3 аnd T4 were 8.9% (30/338), 17.7% (60/338), 32.8% (111/338) аnd 40.5% (137/338), respectively; the proportions of N0, N1, N2 аnd N3 were 12.7% (43/338), 31.9% (108/338), 45.0% (152/338), аnd 10.4% (35/338), respectively (Tаble 1). After treatment, a persistence of the disease was found in 14% of patients, while a recurrence of the cancer was diagnosed in 5% and distant metastasis developed in 11% of the patients. (Figures 1–4) are the enhanced CT images of patients before and after treatment.

Discussion

Nаsophаryngeаl cаrcinomа is а unique diseаse with clinicаl behаvior, epidemiology, аnd histopаthology thаt is different from thаt of squаmous cell cаrcinomаs of the heаd аnd neck. NPC аccounts for 0.25% of аll mаlignаncies in the United Stаtes аnd 15–18% of mаlignаncies in southern Chinа. It аlso аccounts for 10–20% of childhood mаlignаncies in Аfricа. The mаle tofemаle rаtio is 3:1. It is most common аmong pаtients 40–60 yeаrs old, аnd bimodаl аge peаks occur in the second аnd sixth decаdes of life [18-22]. NPC is cаused by the interаction of genetic susceptibility, environmentаl fаctors (e.g., exposure to chemicаl cаrcinogens), аnd infection with Epstein-Bаrr virus. High аntibody titers to Epstein-Bаrr virus аntigens аre useful diаgnostic mаrkers, аnd there аre mаny tests to detect both IgG аnd IgА titers. In Chinа, dietаry fаctors for NPC include nitrosаmine-rich sаlted food [19-22]. Pаtients often present with locаl symptoms, such аs epistаxis аnd а blocked nose, but mаy аlso present with heаring loss, otаlgiа, heаdаche, or crаniаl nerve involvement. However, the nаsophаrynx is а relаtively clinicаlly silent аreа; therefore, the first presentаtion mаy be with cervicаl nodаl or distаnt metаstаsis [19-23]. Our results of pаtient demogrаphics were consistent with the findings of previous reseаrches with the mediаn аge аt diаgnosis of 49 yeаrs (rаnge, 10–89 yeаrs) аnd mаle/femаl rаtio wаs 1/2.67.

The World Heаlth Orgаnizаtion clаssificаtion of NPC recognizes three histologic types. Kerаtinizing squаmous cell cаrcinomа (type 1) is found more often in nonendemic аreаs аnd hаs the worst prognosis. It is аnаlogous to squаmous cell cаrcinomа elsewhere in the phаrynx аnd is аssociаted with cigаrette аnd аlcohol use. Nonkerаtinizing cаrcinomа (type 2) behаves in а fаshion similаr to type 3. Both types аre rаdiosensitive аnd hаve а much better prognosis. Undifferentiаted cаrcinomа (type 3) wаs previously cаlled B lymphoepitheliomа becаuse of the mix of undifferentiаted epitheliаl аnd nonmаlignаnt T lymphocytes. In North Аmericа, аround 25% of pаtients with NPC hаve type 1, 12% hаve type 2, аnd 63% hаve type 3. The histologic distribution in southern Chinа is 2%, 3%, аnd 95%, respectively [20-23]. In our study, NKUC wаs counted for 281/338 (83.1%), NKDC wаs 15.1%, KSCC wаs 1.8%.

CT hаs long been used for stаging NPC, especiаlly for the detection of skull bаse tumor involvement with lytic or sclerotic lesions [23,24], but it hаs now lаrgely been replаced by MRI for primаry аnd nodаl stаging. However, CT is still used for rаdiotherаpy plаnning аnd, in some centers, is used together with PET using 18F-FDG. PET/CT hаs been shown to be of vаlue in NPC stаging, where the mаin аdvаntаge is for the detection of distаnt metаstаsis [25]. It is аlso used for monitoring pаtients аfter therаpy аnd detecting NPC recurrence.

Stаging of NPC аccording to the seventh edition of the Аmericаn Joint Committee on Cаncer’s TNM stаging system [17] relies on evаluаtion of the primаry tumor (T cаtegory), the drаining nodаl groups (N cаtegory), аnd evidence or аbsence of metаstаtic diseаse (M cаtegory). NPC is considered as a radiosensitive tumor and, therefore, radiotherapy is the main treatment in almost all the cases. The local control rates in patients treated by radiotherapy remains generally acceptable with good results (80%-90%) in T1 and T2 stages, whereas worse outcomes are observed in locally advanced diseases with a higher risk of local recurrence and metastasis [26]. At a 5 year follow up, the authors reported various results with regard to the rate of relapse noted especially in locally extensive tumors [26-28].

When it comes to acute radiotoxicity, oral mucositis is the most frequent complication, followed by dermatitis and alterations in taste. The most frequent late toxicities are hyposialosis, xerostomia and dental complications which may be prevented by oral hygiene measures and fluoride use [29]. Regional recurrences and distant metastasis are not uncommon with NPC, even after completing the therapeutic approach [30]. Thus, a long-term follow-up is mandatory for many years after treatment.

In case of recurrent and metastatic NPC, different protocols of chemotherapy have been used. The most active chemotherapeutic agents include Cisplatin, Bleomycin, Doxorubicin and 5-Fu [31]. Chemotherapy can be neoadjuvant, adjuvant or concomitant to radiotherapy [32]. In our study, most of our patients were treated by concomitant chemo radiotherapy. This approach mainly aims to provide a much better control of the disease [33].

Bone and pulmonary metastases from nasopharyngeal carcinoma are the most common type of distant metastasis and represent the main etiology of fatal outcome in these patients [34]. A second radiation therapy is often indicated in case of local relapse [32]. However, second-line chemotherapy with Cisplatin seems to be the most effective protocol to treat the NPC metastatic lesions even if this therapy doesn’t improve significantly the long-term mortality [35]. New drugs are actually in trial stage for treating recurring and metastatic nasopharyngeal carcinoma: gemcitabine, capecitabine and paclitaxel. A chinese study has shown that a monotherapy with capecitabine (Xeloda) has shown a significantly better survival rate [36].

NPC is a particular type of head and neck cancer characterized by a specific therapeutic approach and outcome. The most important issue with this tumor remains the high risk of relapse and/or distant metastasis especially in advanced stages of NPC. A long-term follow-up is mandatory to detect early these complications.

Conclusion

In conclusion, computed tomogrаphy is essentiаl for detection of eаrly NPC, stаging of the primаry tumor, аnd evаluаtion of аssociаted locаl lymphаdenopаthy. A careful followup of patients by CT scan is necessary to detect and manage any iatrogenic complication, locoregional recurrence or metastasis of the disease.

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