Effect of Ultrasonic Cleaning of Titanium Turning Scraps Immersed in Alkaline Solution and Subsequent Preparation of Ferrotitanium Ingots
2021

Suhwan Yoo
†
Jikwang Chae
Jung-Min Oh
Jae-Won Lim
The Research Institute of Mineral Resources and Energy Development
Jeonbuk National University
54896JeonjuRepublic of Korea

Division of Advanced Materials Engineering
College of Engineering
Jeonbuk National University
54896JeonjuRepublic of Korea

Effect of Ultrasonic Cleaning of Titanium Turning Scraps Immersed in Alkaline Solution and Subsequent Preparation of Ferrotitanium Ingots

Korean J. Met. Mater.)
592202110.3365/KJMM.2021.59.2.113(Received August 13, 2020; Accepted December 29, 2020)Ti scrapspretreatmentrecyclingultrasonic cleaningferrotitanium
Ti has excellent properties but is more expensive than other materials due to its high melting point, high reactivity and difficult processability. One way to lower the production cost of Ti products is to recycle Ti scraps. Before recycling Ti scraps, pretreatment to remove contamination is essential. Ti scraps can be pretreated in an alkaline solution, thus eliminating the use of acidic solutions. However, a pretreatment only involving immersion in alkaline solution requires a high concentration solution, and has low efficiency. Therefore, in this study, an optimized ultrasonic cleaning process is introduced to pretreat Ti scraps in a lowconcentration alkaline solution. The carbon content of Ti scraps before pretreatment was 6,800 ppm, and showed a sharp decrease with pretreatment. Using this pretreatment process, C, O, and N impurities were removed by 97.6%, 58.8%, and 29.2%, respectively. Ferrotitanium ingots were then produced by vacuum arc melting (VAM) and plasma arc melting (PAM) using the pretreated Ti scraps and electrolytic iron. Differences in ingots melted by VAM and PAM were investigated. As the time was increased, the content of impurities decreased. Subsequently, a ferrotitanium ingot prepared using the pretreated Ti scraps showed that the carbon concentration in the ingot was less than 200 ppm.

INTRODUCTION

Ti and Ti alloys are high-temperature and light-weight metals with excellent properties, such as high strength, toughness, high degree of corrosion resistance, and biocompatibility. Owing to these characteristics, Ti and Ti alloys are used for light-weight components not only in the aerospace industry, but also in high performance applications, such as automobiles and implants [1][2][3][4]. However, the production of Ti-components entails high cost because of the metal's high reactivity with oxygen and atmospheric gases [5]. Since Ti has a particularly high oxygen affinity, the manufacturing process requires considerable amounts of energy, and results in large quantities of scrap. This consequently raises the price of Ti products. Therefore, a large number of researches studies have been conducted on the recycling of Ti scraps [6][7][8][9].

114

대한금속 · 재료학회지 제59권 제2호 (2021년 2월) contaminated by cutting oil. Cutting oil provides cooling and lubrication properties. However, the used cutting oil accumulates physical and chemical contaminants, losing it properties and becoming contaminated, producing toxic waste [13][14][15][16][17]. Therefore, raw scraps have a harmful effect on the product. When using Ti scraps to obtain a Ti alloy with desired characteristics, a pretreatment process to remove the cutting oil is required.

W2 types of water-soluble cutting oil are mainly used for titanium cutting. Since the surface temperature of the cutting process is not high enough to form TiC, the remained cutting oil on the surface is retained during recycling, and has a greater effect on product properties. If the oil on the surface of the Ti scraps is not completely removed, it becomes a major source of gaseous impurities during melting and recycling. In particular, the adhered oil remains as carbon impurities in the molten metal during the subsequent melting process. An acidic solution or organic solvent is generally used to remove not only the oil, but also the oxygen layer. In addition, this also improves porosity [18][19][20][21][22]. However, this process may pose problems to the environment and process safety.

To address this issue, a relatively eco-friendly alkaline degreasing solution, such as an aqueous solution based on NaOH, is used [23,24]. Alkaline degreasing mainly involves a saponification reaction between the oil and the aqueous alkaline solution. Saponifiable fats react with the aqueous alkaline solution, dividing it into saponin and glycerin, which are sodium fatty acids. Saponins are subjected to emulsification and dispersion by the aqueous alkaline solution.

In our previous study [25], Ti turning scraps were immersed in an alkaline solution to avoid the use of an acidic solution.

However, the concentration of the alkaline solution had to be very high. Hence, in this study, to reduce the concentration of the solution, we conducted ultrasonic cleaning of the Ti scraps immersed in an alkaline solution. The optimization of the alkaline degreasing solution for ultrasonic cleaning of the contaminated Ti turning scraps was carried out. Subsequently, we also investigated the effect of the ultrasonic cleaning process on the preparation of ferrotitanium ingots using the pre-treated Ti turning scraps.

EXPERIMENTAL PROCEDURES

The overall flow of the experiments is shown in Fig. 1. The solution for the pre-treatment of Ti scraps was primarily composed of NaOH or tetrasodium pyrophosphate (TSPP; Na 4 P 2 O 7 ) based on a previous study [25]. However, to pretreat Ti scraps in lower concentrations of the solution, ultrasonic cleaning was adopted, which demonstrated good efficiency in cleaning [26]. Ultrasonic cleaning creates shock Next, ferrotitanium ingots were prepared by melting the pre-treated Ti scraps along with electrolytic iron at a mass ratio of 7:3. Twenty-one g of Ti and nine g of Fe were pressed under 45 MPa pressure in a compaction mold with a diameter of 30 mm, to form a green compact. Thirty g of this green compact was placed on a water-cooled copper mold in a chamber, and the chamber was evacuated to an air pressure of 5.5 × 10 -3 torr. Next, ultra-high-purity argon (99.999%) was added to the chamber to bring its pressure back to atmospheric pressure. Then, the green compact was premelted by an electrical discharge at an output of 5 kW. The ferrotitanium ingots were prepared using two methods, vacuum arc melting (VAM) and plasma arc melting (PAM).

The melting times with these two methods were 10 and 20 min, respectively. X-ray diffraction (XRD) analysis of the ferrotitanium ingots was performed (SHIMAZU, LabX XRD-6100, 40 kV, 30 mA, Cu-Ka radiation). After etching for 3 seconds in a solution (water : nitric acid : hydrofluoric acid = 97 : 2 : 1), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) were used to observe the surface of the ingots and analyze their microstructure.

RESULTS AND DISCUSSION

There have been many reports on the subject of cleaning oil contaminants [27][28][29]. Categories of oil washing can be classified into roll-up, emulsification, and solubilization mechanisms. Depending on the interaction between the solution, oil, and substrate, the contact angle and mechanism are different, and can be expressed through the following equation.

where γ ow is the interfacial tension between the solution and the oil; γ os is the interfacial tension between the oil and the substrate; γ ws is the interfacial tension between the solution and the substrate, and θ is the contact angle. When γ os is greater than γ ws , θ will be greater than 90 o . Under these conditions, the roll-up mechanism is easy to operate. In this mechanism, the solution wets the substrate and removes the oil. A schematic diagram of the roll-up mechanism is shown in Fig. 2(a).

On the other hand, when γ ws is greater than γ os , θ will be smaller than 90 o , and the emulsification mechanism is more effective. Here, the oil is not completely removed, and necking occurs as shown in Fig. 2(b). Finally, the third mechanism, namely direct solubilization, shown in Fig. 2
(c),
can operate when there is enough surfactant in the solution [28].

There is, however, another mechanism for forming an According to an earlier study on the cleaning of oils on substrates [27], the contact angle and wettability of the oil depend on the pH of the solution, and the Ti scraps tend to be washed well at a pH of 9.5−10.5 [29]. The compositions of the pre-treatment solutions considered in this study (designated as PT-1 through PT-5), in the above-mentioned pH range, are shown in Table 1 and oil removal effect when washing cotton fabrics. Also, pyrophosphate gives greater lathering effect [31,32]. In previous studies, cleaning was carried out at a relatively high concentration to remove the oil from Ti scraps [25].

However, through a salting-out effect on the soap, hydroxyl ions reduce the surface activity [31]. To improve this, variables were applied to the NaOH and TSPP concentration.

Here, the concentrations of PT-1 and PT-4 were adjusted using NaOH, while those of PT-2, PT-3, and PT-6 were adjusted using TSPP. PT-5 consists of NaOH and TSPP. In addition, to study the effect of ultrasonic cleaning, PT-6 had the same composition as PT-2, but without ultrasonic cleaning.

The degreasing in an alkaline aqueous solution during the pre-treatment can be represented by the following equation.

From the chemical equation, it is seen that the higher the NaOH concentration, the better the reaction is, because NaOH is the main source of the reaction. In a previous report pertaining to immersion washing [25], it was observed that the degree of washing increased as the concentration was increased up to 2M NaOH. However, during the pretreatment using ultrasonic cleaning, the solution containing large amounts of NaOH tended to wash the Ti scraps to a lesser extent.

Comparing the amount of C remaining in PT-1 and PT-4 in Table 2, it can be seen that PT-4, which has a higher NaOH concentration was washed less than PT-1. However, it may be also observed that the reaction of NaOH was not the main mechanism of washing. On the contrary, the washing was predominantly a function of the contact angle and the wettability, which were in turn, affected by the pH [27].

Thus, if the pH is very high, then the contact angle becomes small, and hence, it can be inferred that the oil remains on the substrate by the emulsification mechanism, and not the rollup mechanism. PT-2, PT-3, and PT-6, which were composed of TSPP, showed lesser change in pH after washing compared to PT-1, which was composed of NaOH. They confirmed that the cavitation from ultrasonic affected the results.

In the case of PT-1 and PT-4, NaOH reacted with the oil, resulting in a lower pH. But in the case of PT-4, there was very little pH change because the NaOH concentration was too high. On the other hand, in the case of PT-2 and PT-3, Na + in TSPP was dissociated, adjusting the pH to a proper level, and also acted as a buffer, which suppressed the change in the pH value. Compared to PT-2, PT-5 had more carbon left. This shows that pH, even in the presence of TSPP, affected the contact angle and surface activity.       large amount of C in ferrotitanium, its performance as a deoxidizer tends to be poor [11,12].

Next, the effect of melting method on ferrotitanium production using the Ti scraps was investigated. The PT-2 scraps, which showed the highest oil removal effect, were used to investigate the change in gas impurities in the ingots prepared by VAM and PAM, as a function of melting time.

The variations in C, O, and N with the melting time of the ferrotitanium ingots prepared by the two different melting methods are shown in Fig. 6 and Table 3. As the melting time of VAM and PAM was increased, the contents of C, O, and N in the ferrotitanium ingots decreased. However, it was confirmed that the gas impurities in the ferrotitanium ingots melted by PAM, in general, had lower levels of concentration.

The removal degree, RD (%) of each gas impurity was defined by the following equation.

(3)   where C i and C f are the initial and final concentrations. The overall removal degree RD av (%) of the gas impurities is defined as (4) PAM tends to be slightly higher in RD av (%) than VAM.
RD % ( ) 100 C i C f - ( ) =
This is because high energy density could be seen in PAM, owing to the shrinkage nozzle and the orifice gas [30].

SEM and EDS images of the distribution of Ti, Fe, and C in the ferrotitanium ingot melted by PAM for 20 min are shown in Fig. 7. As can be seen from the maps, (d), Fe was distributed in the matrix; however, it was distributed more in the precipitated phase. On the other hand, in image (e), Ti was more distributed in the matrix. This was consistent with the distribution of Fe when the matrix phase was β-Ti and the precipitation was TiFe. In addition, from the map in (c) it can be confirmed that a carbide phase containing a significant amount of C was not observed.

In this study, the pre-treatment process was optimized by ultrasonic cleaning the oil-stained Ti turning scraps in a low concentration alkaline degreasing solution. Carbide was not observed in the ferrotitanium ingots made of pre-treated scraps obtained from the optimized pre-treatment process, and the ingots revealed a eutectic structure composed of β-Ti and TiFe. In addition, the ferrotitanium ingot prepared by PAM showed lower concentration of gaseous impurities than that made by VAM.

CONCLUSIONS

In this study, Ti turning scraps were cleaned using an optimized pre-treatment process. In the pre-treatment process, ultrasonic cleaning was performed with low alkaline concentration solutions. Ferrotitanium ingots were prepared by VAM and PAM to compare the relative reduction in gaseous impurities. The results obtained can be summarized as follows:

(1) Ultrasonic cleaning of Ti scraps immersed in a low concentration alkaline solution was used to remove the oil on the surface of the Ti turning scraps.

(2) At an appropriate pH, the contact angle increased and the oil became spheroid, and when the pH value was high, the contact angle decreased and became oil wet.

(3) For solutions with higher pH, cleaning efficiency was reduced -the roll-up mechanism had a greater effect on cleaning than the solubilization mechanism.

(4) The optimum pretreatment composition for the ultrasonic cleaning of Ti scraps was found to be a 10 g/L TSPP (Na 4 P 2 O 7 ) solution with appropriate pH (9.66).

(5) The oil was easily separated from the solution containing TSPP, but the results showed that the removal was better at the proper pH.

(6) After ultrasonic cleaning using the optimum conditions, the removal degrees of C, O, and N gaseous impurities were 97.6%, 58.8%, and 29.2% respectively.

(7) A ferrotitanium ingot of less than 200 ppm carbon was prepared by PAM.

waves by forming micropores in the solution and its cleaning effect is based on creating random or irregular agitation in the solution. To remove the oil adhering to the surface of the Ti scraps, they were immersed in the alkaline solution, washed with an ultrasonic cleaner at 40 kHz for 10 min, and then rinsed. The washed scraps were dried at 70 o C for 2 hr in a vacuum oven to produce pre-treated scraps. An industrial digital camera (SKT ML500C-125A) was used to photograph the contact angle. The pH of the solution before and after washing was measured by a pH meter (Mettler Toledo, Five Easy Plus. Carbon, oxygen, and nitrogen analyses (Eltra CS-2000, ON-900) were performed to determine the concentration of gas impurities in the Ti scraps.

Fig. 1 .
1Flowchart of the process and photographs of the materials: (a) Ti turning scraps, (b) pure electrolytic Fe, and (c) ferrotitanium ingot.

intermediate phase at the interface between the solution and the oil. This mechanism operates when the oil contains a lot of polar components. When the liquid crystals have grown sufficiently through the interaction of polar oil with the adsorbed surfactant, the intermediate phase is decomposed by stirring and falls off into solution.

Fig. 2 .
2. TSPP is a representative component used as soap builder and has the appropriate pH Oil removal mechanisms: (a) roll-up, (b) emulsification, and (c) solubilization.116대한금속 · 재료학회지 제59권 제2호 (2021년 2월)

Figure 3
3is an image taken over time after sessile drop, used to observe the contact angle. The substrate was prepared by polishing after melting the Ti scraps, and the oil was obtained from the surface of the Ti scraps.Figure 3(a)~(c) and (g)~(i) are solutions consisting only of NaOH. In the case of PT-1, oil was spheroidized over 15 seconds by increasing RCOOR' NaOH RCOONa R'OH 23 [ ] + → +

angle, and there was no significant change afterwards. On the other hand, with PT-4 the contact angle became smaller and oil wet. It was confirmed that this difference in contact angle was due to the pH value. In the compositions PT-2 and PT-3 containing TSPP, the oil was spheroidized and then fell off as in Fig. 3(d)~(f). The gas impurities in the Ti scraps before and after pretreatment are shown in Fig. 4. The gas impurities were mostly contained in cutting oil adhered on the surface of scraps, otherwise it would be removed by deterioration during processing. The carbon concentration showed the biggest change after pre-treatment, and the Ti-scraps pretreated with PT-2 were the best washed. The amounts of C, O, and N in the raw scraps were 6,820, 2,355, and 705 ppm, respectively. After the pre-treatment with PT-2, these impurities were reduced to 195, 970, and 325 ppm, respectively. The high reductions with PT-2 may be attributed to the fact that the oil on the surface of the scraps was separated better owing to the wettability and contact angle, which in turn, were due to the proper pH value of PT-2.

Figure 5
5shows the results from the XRD analyses of ferrotitanium ingots prepared with both the Ti raw scraps and the PT-2 scraps. This figure demonstrates the effect of oil on

Fig. 3 .
3Photograph of oil shape by solution and time: (a) ~ (c) PT-1, (d) ~ (f) PT-2, (g) ~ (i) PT-4.

Fig. 4 .
4Comparison of gaseous impurities in Ti turning scraps after pre-treatment with PT-1 -PT-4.

Fig. 5 .
5XRD patterns of ferrotitanium ingots prepared by raw and PT-2 scraps.대한금속 · 재료학회지 제59권 제2호 (2021년 2월)the formation of the ferrotitanium ingots. The ingots were pre-melted with Ti scraps and electrolytic iron at a 7:3 mass ratio. The peaks observed inFig. 5were mainly β-Ti, α-Ti and TiFe. The XRD peaks of PT-2 were divided into two peaks around 40 degrees, which were β-Ti and α-Ti. In the case of pre-melting, an α-Ti peak was observed because electrolytic Fe, which is a β-Ti stabilizing element, could not contribute to homogenization, due to insufficient melting time. The XRD pattern of the ferrotitanium ingot made of raw scraps showed a TiC peak, which was absent in the ingot made of PT-2 scraps. This difference could be attributed to the large amount of C contained in the oil. When there is a

Fig. 6 .
6Concentration variation of gaseous impurities in the ferrotitanium ingots refined by VAM and PAM as a function of melting time.

Fig. 7 .
7SEM images and EDS analyses of ferrotitanium ingot made by PAM: (a) microstructure, (b) EDS mapping image, (c) C, (d) Fe, and (e) Ti.

Table 1 .
1Experimental conditions of pre-treatment process.Pre-treatment
Type
Solution concentration
pH Before
pH After
PT-1
Ultrasonic
NaOH 3.2 × 10 -5 M
9 . 5
7 . 6
PT-2
Ultrasonic
Na 4 P 2 O 7 10 g/L
9.66
9.64
PT-3
Ultrasonic
Na 4 P 2 O 7 20 g/L
10.00
9.97
PT-4
Ultrasonic
NaOH 1 M
14
14
PT-5
Ultrasonic
NaOH 1M + TSPP10 g/L
14
14
PT-6
Immersion
Na 4 P 2 O 7 10 g/L
9.66
9.65

Table 2. Change in the concentrations of gaseous impurities in the
ferrotitanium ingots before and after VAM and PAM.

Table 3 .
3Change in the concentrations of gaseous impurities in the ferrotitanium ingots before and after VAM and PAM.%
C
O
N
S u m R D av (%)
pre-melting
175
960
290
1,425
VAR 10 min
170
935
265
1,370
3.86
VAR 20 min
160
890
215
1,265
11.23
PAM 10 min
145
890
255
1,290
9.47
PAM 20 min
135
865
210
1,210
15.09

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Supplement Timing of Cranberry Extract Plays a Key Role in Promoting Caenorhabditis elegans Healthspan
2014

Sujay Guha
Department of Biological Sciences
Clemson University
29634ClemsonSCUSA

Ojas Natarajan
Department of Biological Sciences
Clemson University
29634ClemsonSCUSA

Cole G Murbach email@example.com.
Department of Biological Sciences
Clemson University
29634ClemsonSCUSA

Jessica Dinh firstname.lastname@example.com.
Department of Biological Sciences
Clemson University
29634ClemsonSCUSA

Ethan C Wilson
Department of Biological Sciences
Clemson University
29634ClemsonSCUSA

Min Cao email@example.com.
Department of Biological Sciences
Clemson University
29634ClemsonSCUSA

Institute for Engaged Aging
Clemson University
29634ClemsonSCUSA

Sige Zou email@example.com
Functional Genomics Unit
Translational Gerontology Branch
National Institute on Aging
21224BaltimoreMDUSA

Yuqing Dong
Department of Biological Sciences
Clemson University
29634ClemsonSCUSA

Institute for Engaged Aging
Clemson University
29634ClemsonSCUSA

Supplement Timing of Cranberry Extract Plays a Key Role in Promoting Caenorhabditis elegans Healthspan

Nutrients
6201410.3390/nu6020911Received: 25 November 2013; in revised form: 20 December 2013 / Accepted: 11 February 2014 /nutrients † These authors contributed equally to this work. * Author to whom correspondence should be addressed; E-Mail: email@example.com;aging interventiontimingnutraceuticalscranberry polyphenolshealthspanCaenorhabditis elegans
Consumption of nutraceuticals is a major and potent dietary intervention for delaying aging. As the timing of administration is critical for the efficacy of bioactive compounds in medicine, the effectiveness of nutraceuticals may also be dramatically affected by the timing of supplementation. Cranberry exact (CBE), rich in polyphenols, is consumed as a nutraceutical, and possesses anti-aging properties. Here, we examined the influence of timing on the beneficial effects of CBE supplementation in C. elegans. The prolongevity effect of CBE in different aged worms, young adults, middle-age adults, and aged adults, was determined. Early-start intervention with CBE prolonged the remaining lifespan of worms of different ages more robustly than late-start intervention. The effectiveness of CBE on stress responses and physiological behaviors in different aged worms was also investigated. The early-start intervention prominently promoted motility and resistance to heat shocks and V. cholera infection, especially in aged worms. Together, these findings suggest that the timing of CBE supplementation critically influences itsOPEN ACCESSNutrients 2014, 6 912 beneficial effects on C. elegans lifespan and healthspan. It is of interest to further investigate whether the similar results would occur in humans.

early developmental stage played a more critical role in CBE-mediated prolongevity relative to CBE long-term treatment. We further examined worms' responses to heat shock and V. cholerae infection and their physiological locomotion using similar timing strategies. Our results similarly indicated that the early-start intervention leads to more prominent benefits to C. elegans overall health status as compared to the late-start intervention. Briefly, the CBE early-start intervention significantly improved their motility and heat shock tolerance, especially in aged worms. The early-start intervention, not the late-start intervention, has the significant efficacy in preventing V. cholerae infection in C. elegans. Taken together, our findings reveal that CBE supplementation starting at the early developmental stages is critical to optimizing its benefits in C. elegans healthspan.

Materials and Methods

CBE Preparation

The CBE, rich in polyphenols, kindly provided by Dan Souza (Decas Botanical Synergies, Carver, MA, USA), was described in a previously published study [14]. To prepare cranberry supplemented food, the CBE in powder was first dissolved in sterile distilled water. A fresh 10% (w/v) stock solution was prepared one day before the assay and then the appropriate dilutions were overlaid onto nematode growth medium (NGM) plates.

C. elegans Maintenance

Wild type N2 worms were obtained from the Caenorhabditis Genetics Center (CGC), University of Minnesota, Minneapolis, MN, USA and maintained at 20 °C on NGM seeded with E. coli OP50 feeding strain. A volume of 100 µL of OP50 (overnight culture) was dropped on the center of 60-mm NGM plates, which were allowed to dry overnight before worms were transferred.

Lifespan and Remaining Lifespan Assay

All assays of lifespan and remaining lifespan were carried out at 25 °C. Synchronized populations were obtained by allowing 10-15 hermaphrodites lay eggs overnight at 16 °C onto E. coli OP50 seeded regular NGM plates or treatment plates (NGM plates containing 2 mg/mL CBE), and then the parents were removed the next day. The eggs were allowed to hatch and develop until reaching L4 stage.

To carry out early-start intervention with CBE, synchronized L4 stage worms from treatment plates were transferred onto a modified treatment plate (treatment plate containing 50 μg/mL FUDR to prevent the growth of progeny) seeded with E. coli OP50 and then continue to grow for 2 days, 6 days, and 10 days, respectively. For the late-start intervention with CBE, synchronized L4 stage worms from regular NGM plates were transferred onto a modified treatment plate seeded with E. coli OP50 and continue to grow for 2 days, 6 days, and 10 days, respectively.

For remaining lifespan assays, 30 worms from each time point after either early-start intervention or late-start intervention, were transferred to the FUDR-containing NGM plates and remaining lifespan assays were carried out until all worms died. Worms of the same ages from regular NGM plates without CBE intervention were transferred onto a FUDR-containing NGM plate as controls. The dead worms were counted starting the day after worms finished CBE intervention and exploding, protruding, bagging or contaminated worms were censored if applicable. We defined the day when the worms finished early-start intervention and late-start intervention as day 0 of the remaining lifespan. E. coli OP50 used for remaining lifespan assays was 10-fold concentrated from overnight culture by centrifugation.

For early-start intervention mediated lifespan assays, 30 L4 stage worms from treatment plates were transferred onto a modified treatment plate seeded with E. coli OP50 to begin the lifespan assay. Leftover worms after 2 days, 6 days, and 10 days, were, respectively, transferred to the FUDR-containing NGM plates without CBE, and the lifespan assays continued until all worms died. Synchronized L4 worms from regular NGM plates were transferred onto the FUDR-containing NGM plates as controls. The dead worms were counted starting the next day after L4 worms were transferred and exploding, protruding, bagging or contaminated worms were censored if applicable. We defined the day when we transferred L4 worms as day 0 of the lifespan. E. coli OP50 used for lifespan assay was 10-fold concentrated from overnight culture by centrifugation.

All the assays were carried out in triplicates and a minimum of two independent trials were performed for all conditions. All statistical analyses were carried out using SPSS software (IBM SPSS Statistics, Armonk, NY, USA). Kaplan-Meier lifespan analysis was carried out, and p values were calculated using the log-rank test, p < 0.05 was accepted as statistically significant. The significant different groups remained statistically significant at p < 0.05 after Bonferroni corrections for multiple comparisons (data not shown).

Heat Shock Assay

Synchronized worms, after either early-start intervention or late-start intervention, were harvested and transferred to regular NGM plates. Worms on regular NGM plates without CBE intervention served as controls. The plates were incubated at 37 °C for 1.5 h and then transferred back to 25 °C. The survival of the worms was monitored daily until all worms died. Each assay was carried out in three independent trials, and the mean survival time (lifespan) was analyzed using SPSS software (IBM SPSS Statistics). Kaplan-Meier lifespan analysis was carried out and p-values were calculated using the log-rank test, p < 0.05 was accepted as statistically significant. On the basis of lifespan assays, the percentage increases of mean survival time were calculated and pooled.

Bacterial Killing Assay

V. cholerae killing assays were carried out by performing the remaining lifespan assay on FUDR-containing NGM plates seeded with V. cholerae wild type C6706 (O1 Biotype El Tor strain), rather than OP50. V. cholerae used for killing assay was 10-fold concentrated from overnight culture by centrifugation.

Motility Assays

Ten CBE-treated (early intervention and later intervention) or non-treated day 2, day 6, and day 10 adult nematodes were placed onto individual NGM plates without OP50. The body bends per minute were counted. This assay was carried out in three independent trials. The data were pooled and analyzed using Student's t test, p < 0.05 was accepted as statistically significant.

Results and Discussion

Early-Start Intervention with CBE Shows More Potency in Increasing the Remaining Lifespan of Elderly Worms Relative to the Late-Start Intervention

To determine the best timing of CBE supplementation, we compared the prolongevity efficacy of early-start intervention and late-start intervention in different aged worms. The early-start intervention initialized the CBE treatment prior to eggs hatching, and continued until reaching young adult, middle-age adult, and aged adult. The late-start intervention began the CBE treatment from L4 stage and continued to young adult, middle-age adult, and aged adult. We summarized the intervention methods in Figure 1. Thereafter, we measured the remaining lifespan of young adult, middle-age adult, and aged adult, respectively. For the early-start intervention to young adult worms, the remaining lifespan was prolonged from 10.1 days to 11.2 days, which increased 11.1% (Table 1), while the late-start intervention prolonged the remaining lifespan of young adults from 10.1 days to 10.9 days, an increase of only 8.2% (Table 1). A 32.7% increase of remaining lifespan was observed, in the early-start intervention to middle-age adults, while the late-start intervention increased 21.2% (Table 1). Impressively, the early-start intervention to aged adults resulted in an 80.8% increase in remaining lifespan, and the late-start intervention led to a 69.7% increase (Table 1). Our data indicated that although the late-start intervention could extend the remaining lifespan in different aged worms, the early-start intervention overall showed greater efficacy. Most notably, our data clearly showed that CBE supplementation lasting until the worms were aged dramatically elevated the remaining lifespan of the aged population, especially the early-start intervention (Table 1). These findings suggest that in order to fully exert CBE's prolongevity effects, CBE supplementation should start in the early developmental stage and be continued in a long-term manner.

Early Developmental Stages of C. elegans Play More Prominent Role in CBE-Mediated Lifespan Extension as Compared to CBE Long-Term Supplementation

As both of the early developmental stage and long-term treatment contributed to the CBE-mediated prolongevity, we next wanted to assess and compare their prolongevity potency. To this end, we carried out a regular lifespan assay to measure the effectiveness of each early-start intervention method (Table 2, Figure 2). Compared to untreated worms, C. elegans treated with CBE from egg until young adult, increased the mean lifespan of worms from 12.5 days to 14.1 days, resulting in 12.8% increase. The early-start intervention to middle-age prolongs the mean lifespan to 14.3 days (14.4% increase), and the early-start intervention to aged adult prolongs the mean lifespan up to 15.0 days (20.0% increase). Considering that the larval stage from egg to L4 only takes ~1.5 days (36~40 h) at 25 °C, the early-start intervention to young, middle-age, and aged adults takes around 3.5, 7.5, and 11.5 days, respectively. Compared to the 12.8% increase (intervention to young adults), the intervention to aged adults took eight more days but only contributed an additional 7.2% increase (20% increase in total). Hence, our results imply that the early developmental stage plays more prominent role in CBE-mediated lifespan extension as compared to CBE long-term supplementation. Results presented in Figure 2. The lifespan experiments were repeated at least three times with similar results, and the data for representative experiments are shown. The lifespan data were analyzed using the log-rank test and p-values for each individual experiment are shown. EI represents the early-start intervention. # indicates number.

Figure 2.

Longer time supplementation with CBE results in more prolonged lifespan extension in C. elegans. Wild type N2 worms were supplemented with 2 mg/mL of CBE through the early-start intervention. Each lifespan experiment was repeated at least three independent times with similar results. Quantitative data and statistical analyses for the representative experiments are included in Table 2. EI represents the early-start intervention.

Influence of CBE Early-Start Intervention and Late-Start Intervention on Worms' Heat Shock Resistance and Motility

Considering the correlation between prolongevity and better health conditions, we examined C. elegans heat shock resistance after either early-start intervention or late-start intervention. Heat shock resistance has been coupled with the benefit of CBE-mediated prolongevity [14]. Our results indicate that both early-start and late-start interventions resulted in statistically significant resistance to heat shock in each age group, as compared to the controls without CBE treatment (Figure 3a). In addition, the early-start intervention shows much stronger potency relative to the late-start intervention (Figure 3a), suggesting that the larval stages (early developmental stage) are important for developing CBE-mediated heat shock resistance. We cannot rule out the possibility that the longer time of intervention may also be critical to improving C. elegans heat shock resistance, as for both interventions there was a positive correlation between length of intervention and increased resistance. However, again, considering that the larval stage from egg to L4 only takes ~1.5 days (36-40 h) at 25 °C, the early-start intervention to middle-age adult will take ~7.5 days, while the late-start intervention to middle-age adult takes six days. This 1.5 days of difference led to a resistance increase from 9.3% (late-start) to 61.3% (early-start) (Figure 3a). This comparison clearly implied that the early developmental stage is important for CBE-mediated heat shock resistance. Similarly, the early-start intervention to aged adult will take ~11.5 days. As compared to the late-start intervention to the aged adult (10 days), the 1.5 days of difference led to the resistance increase from 33.8% (late-start) to 76.5% (early-start), which further confirmed our conclusion that the larval stage is important for establishing CBE-mediated heat shock resistance. Figure 3. Outcomes of the early-start intervention and the late-start intervention on C. elegans heat shock resistance and motility. (a) N2 worms after either the early-start intervention or the late started intervention exhibited increased survival after 1.5 h at 37 °C when compared to control worms; (b) only worms after the early-start intervention showed an elevated motility rate in aged population, as compared to control worms. At least triplicate samples were examined for each stress assay. "% increase" indicates the average increase among the multi-replicates and error bars represent the standard deviation. p value was calculated using Student's t-test. * p < 0.05 when compared to control. Each of the stress assays was repeated at least three independent times with similar results. EI represents the early-start intervention; LI represents the late-start intervention.

Motility is one of the key physiological indexes to assess animals' degree of aging. Given the evidence that both of the early-start and late-start interventions to aged worms can dramatically increase the remaining lifespan (Table 1), we postulated that the early-start and the late-start interventions to aged adults would improve locomotion in aged populations. Surprisingly, only the early-start intervention significantly improved the motility of aged worms. The late-start intervention did not benefit aged worms' motility ( Figure 3b).

Taken together, our findings from the heat shock assay and motility assay imply that aging intervention, if started at the early stage of development, may significantly influence healthy aging in aged populations. This is reasonable since the developmental period before adulthood is extremely important in determining an animal's long-term health [17].

Protective Effects of CBE Supplementation on Vibrio cholerae Killing in C. elegans

Bacterial infection is a leading cause of death among the elderly [18]. We found that CBE supplementation could protect worms from V. cholerae infection (unpublished data) and this protection is not due to the antibacterial effects of CBE (data not shown). Here, we determined how CBE may lead to the best protective effects against V. cholerae. We performed the V. cholerae killing assays in different aged worms. All three of the early-start intervention groups were significantly protected against V. cholerae killing (Table 3). Conversely, the late-start intervention did not show the significant protection in the young, middle-age, or aged worms (Table 3). It is known that the developmental stage before adulthood is an essential period for an animal to develop its immune system [19]. C. elegans feeds on bacteria and has evolved and maintained an innate immune system to defend against pathogenic attack [20]. Therefore, the difference of the early-start intervention and the late-start intervention shown on V. cholerae resistance may indicate that CBE helps C. elegans build up a robust innate immunity in the larval stage. Undoubtedly, to support this hypothesis, more worm pathogens need to be tested in further experiments. The V. cholera killing assays were repeated at least three times with similar results, and the data for representative experiments are shown. The lifespan data were analyzed using the log-rank test and p-values for each individual experiment are shown. EI represents the early-start intervention. LI represents the late-start intervention. # indicates number.

Conclusions

We dissected the timing of CBE application in the present studies and our findings revealed the importance of the early developmental stage before adulthood in aging intervention with CBE. The early-start intervention with CBE significantly and dramatically promoted C. elegans healthspan, especially in aged populations. On basis of our experimental evidence, we are the first to suggest how to implement CBE to optimize its prolongevity effects in a multicellular organism. That is, to promote healthspan, CBE supplementation/intervention should start at the early stage of development and, it is better if the intervention is applied in a long-term manner. Considering that CBE-mediated prolongevity and stress responses require evolutionarily conserved mechanisms among diverse species ranging from C. elegans to mammals [14,21,22], our findings have imperative implications for the application of CBE in improving healthspan in higher order organisms, including humans. Noticeably, however, if the similar results would occur in humans still remains unknown.

Figure 1 .
1A scheme of the early-start intervention and late-start intervention.

Table 1 .
1The remaining lifespan of different aged worms at 25 °C. Lifespan and standard error are shown in days.The remaining lifespan experiments were repeated at least three times with similar results, and the data for representative experiments are shown. The lifespan data were analyzed using the log-rank test and p-values for each individual experiment are shown. EI represents the early-start intervention. LI represents the late-start intervention. # indicates number.Treatment
Mean ± SE Median # of Worms Increase% p-Value

Young adult control 10.1 ± 0.3
10.0
118
-
-
EI to young adult
11.2 ± 0.2
11.0
116
11.1%
0.001
LI to young adult
10.9 ± 0.2
11.0
111
8.2%
0.006
Middle-age control
7.0 ± 0.2
7.0
115
-
-
EI to middle-age
9.3 ± 0.3
9.0
117
32.7%
0.001
LI to middle-age
8.5 ± 0.7
9.0
103
21.2%
0.001
Aged adult control
3.1 ± 0.2
3.0
89
-
-
EI to aged adult
5.6 ± 0.2
6.0
87
80.8%
0.001
LI to aged adult
5.2 ± 0.3
5.0
84
69.7%
0.001

Table 2 .
2The lifespan of worms at 25 °C with various early-start interventions. Lifespan and standard error are shown in days.Treatment
Mean ± SE Median # of Worms p-Value
N2 control
12.5 ± 1.0
13.0
179
EI to young adult
14.1 ± 1.2
14.0
141
0.001
EI to middle-age adult 14.3 ± 0.9
14.0
164
0.001
EI to aged adult
15.0 ± 1.2
15.0
146
0.001

Table 3 .
3The survival time of different aged worms after exposed to V. cholera at 25 °C. Lifespan and standard error are shown in days. Mean ± SE Median # of Worms Increase% p-ValueTreatment
Young adult control
7.5 ± 1.1
7.0
213
-
-
EI to young adult
8.3 ± 1.9
8.0
216
11.5%
0.001
LI to young adult
7.7 ± 1.2
8.0
163
-
0.890
Middle-age control
5.5 ± 1.2
5.0
213
-
-
EI to middle-age
6.8 ± 1.2
7.0
197
23.3%
0.001
LI to middle-age
5.5 ± 0.7
6.0
173
-
0.987
Aged adult control
4.5 ± 1.2
4.0
204
-
-
EI to aged adult
5.6 ± 1.1
6.0
169
25.3%
0.001
LI to aged adult
4.6 ± 0.8
4.0
181
-
0.197

© 2014 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 license (http://creativecommons.org/licenses/by/3.0/).
AcknowledgementsThe authors are grateful to members of the Dong laboratory for helpful discussions. We especially thank Dan Souza (Decas Botanical Synergies, Sagamore, MA, USA) for providing cranberry extract and Jun (Jay) Zhu (University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA) for providing the V. cholerae strain. This work was supported by the Creative Inquiry fund at ClemsonConflicts of InterestsThe authors declare no conflict of interest.
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Severe cytokine release syndrome resulting in purpura fulminans despite successful response to nivolumab therapy in a patient with pleomorphic carcinoma of the lung: a case report

Osamu Honjo
Terufumi Kubo
Fumiko Sugaya
Takahiro Nishizaka
Koji Kato
Yoshihiko Hirohashi
Hiroki Takahashi
Toshihiko Torigoe
Severe cytokine release syndrome resulting in purpura fulminans despite successful response to nivolumab therapy in a patient with pleomorphic carcinoma of the lung: a case report
10.1186/s40425-019-0582-4C A S E R E P O R T Open Access
Background: Immune checkpoint inhibitors (ICIs) have provided more options in the treatment of lung cancer. However, ICIs can cause several unfavorable reactions generally referred to as immune-related adverse effects. Case presentation: In this report, we present the case of a 52-year-old woman with successful regression of pleomorphic carcinoma of the lung following nivolumab therapy. She developed purpura fulminans (PF) ultimately resulting in amputation of both lower extremities. Blood tests revealed thrombocytopenia with increased serum soluble IL-2 receptor, ferritin, and triglyceride levels suggesting hemophagocytic lymphohistiocytosis (HLH). In addition, serum A disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13 activity was decreased, suggesting thrombotic thrombocytopenic purpura (TTP). Further detailed analysis revealed severe hypercytokinemia including increased levels of IL-1β, IL-6, IL-10, TNFα, IFNγ, and G-CSF.Conclusion:The severe systemic inflammatory reaction and impaired peripheral circulation in this patient was attributed to excessive immunological effect induced by nivolumab resulting in cytokine release syndrome (CRS). This is the first report of a patient with multiple pathological conditions including HLH, TTP-like condition, and PF presumably arising from ICI-induced CRS. Further accumulating thoroughly investigated cases would lead to better understanding of the disease and development of reliable cancer immunotherapy.

Background

Immune checkpoint inhibitors (ICIs) are promising alternatives in cancer treatment in addition to surgery, chemotherapy, and radiotherapy. The basic mechanism of action of ICIs is releasing the brakes of the immune regulation, which is a crucial negative feedback mechanism for avoiding excessive immune reaction. Therefore, ICI therapy has resulted in outstanding success on the one hand and lethal immune-related adverse effects (irAEs) on the other, which are now becoming a major concern in further development of reliable cancer immunotherapy [1]. Hematologic adverse effects in particular are life-threatening but occur relatively infrequently; 7 cases of hemophagocytic lymphohistiocytosis (HLH) following ICI therapy have been reported so far (4 cases of melanoma and 1 case each of urinary bladder carcinoma, Merkel cell carcinoma, and non-small cell lung carcinoma) [2][3][4][5][6]. There has been only 1 reported case of thrombotic thrombocytopenic purpura (TTP) following ICI therapy with ipilimumab [7].

In this report, we present a case of pleomorphic lung carcinoma with significant anti-tumor response to nivolumab therapy. The patient also developed severe cytokine release syndrome (CRS), resulting in HLH and amputation of both lower extremities because of purpura fulminans (PF) probably induced by a TTP-like condition. This is the first report of such a case.

Case presentation

A 52-year-old woman visited a hospital with complaints of right axillary swelling. Computed tomography (CT) imaging identified a space-occupying lesion in the S2 region of the right lung. On histological analysis, the tumor showed high-grade cytological atypia with poor intercellular cohesion (Fig. 1a). Immunohistochemical analysis revealed that the tumor was positive for pan-cytokeratin, vimentin, and thyroid transcription factor 1, but negative for CD45 and CD30, indicating pleomorphic adenocarcinoma of the lung. Programmed cell death ligand 1 was positive in more than 95% of tumor cells (Anti-PD-L1, Clone 22C3; DAKO, Glostrup, Denmark; data not shown). The tumor was evaluated as cT4N2M0 in accordance with TNM classification of Malignant Tumors 8th edition (Union for International Cancer Control, Geneva, Switzerland). The patient underwent four courses of chemotherapy (nab-paclitaxel plus carboplatin). She had fever, polyarthralgia, and muscle soreness. In addition, leukocytosis and thrombocytosis were detected on laboratory testing (data not Fig. 1 Clinical data and pathological images. a Tumor cells with a pleomorphic appearance but are less cohesive. Hematoxylin and eosin staining; original magnification × 200. b Computed tomography imaging shows a low-intensity lesion measuring approximately 7 cm in S2 of the right lung (left panel). By 4 months after the last nivolumab administration, no distinctive space-occupying lesion is found (right panel). Graphs representing the trends of c platelet count, d LDH and CPK from the time of admission (day 1) to day 16. e Images of the lower extremities at 2 days (left panel), 10 days (center panel), and 2 months (right panel) after the onset of purpura fulminans shown). Based on these clinical features, she was diagnosed as having paraneoplastic syndrome and not an adverse effect of chemotherapy because of paucity of association between symptoms and medication. The patient showed a partial response to chemotherapy based on the Response Evaluation Criteria in Solid Tumors guidelines at which time the tumor was 70 × 55 × 48 mm in size ( Fig. 1b; left panel). Then, she was treated with four courses of 120 mg (3 mg/kg) of nivolumab every 2 weeks. Although the patient still had the same symptoms that was diagnosed as paraneoplastic syndrome before, there was no newly developed prodrome or mild noticeable irAEs until the last administration of nivolumab. In addition, laboratory tests performed in each hospital visit did not show any remarkable abnormality.

However, 14 days after the last nivolumab administration, she was admitted to our hospital with complaints of asthenia. The patient felt sick only one day before the admission. She had a fever of 38.6°C. There was livedo reticularis on the extremities with systemic purpura. Laboratory results on admission are shown in Table 1. The patient had no relevant past medical history or family history including autoimmune disease. In addition, autoantibodies, including antinuclear antibodies, proteinase 3, myeloperoxidase-antineutrophil cytoplasmic, and anticardiolipin antibodies, as well as multiple aminoacyl-tRNA synthetases or anti-CL-β2GP1 antibodies, were not detected. Culture and microscopy of the blood smear did not detect bacterial infection. Conversely, significantly increased serum levels of IL-1β, IL-6, IL-10, TNFα, IFNγ, and G-CSF indicated a state of cytokine storm. Thrombocytopenia and increased serum soluble IL-2 receptor, ferritin, and triglyceride levels would imply possible HLH. Impaired liver function with increased lactate dehydrogenase and creatine phosphokinase together with exacerbated muscle soreness suggested rhabdomyolysis. The activity of A disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13 (ADAMTS13) was decreased but not less than 10%, which is a criterion for TTP. There was no evidence of infection on blood culture.

Immediately after admission to the intensive care unit, pulse steroid therapy with methylprednisolone 1000 mg/ day for 3 days and systemic prednisolone administration (50 mg/day) were instituted. Thrombomodulin and mycophenolate mofetil was administered thereafter. Any of them could not provide a dramatic amelioration of her general condition, although the laboratory test showed improvement after administration of thrombomodulin and mycophenolate. Trend of platelet count, blood concentration of lactate dehydrogenase and creatine phosphokinase, which were useful as indicator of the clinical state, are depicted in Fig. 1c and d. By 3 days after hospitalization, general hypokinesis of the left ventricular wall resulted in reduced ejection fraction (20%) and aggravation of respiratory distress. Although we did not perform myocardial biopsy, this was probably caused by CRS induced myocarditis, as evidenced by increased levels of the cardiac marker creatine kinasemuscle/brain in the circulation. Continuous hemodiafiltration was also initiated because of renal failure.

By 7 days after hospitalization, the lower extremities were turning black due to circulatory failure (Fig. 1e). Gangrene then spread with infection involving the entire lower extremities, suggesting PF. Amputation of the left and right lower limbs was performed at three and four months after admission, respectively. We could not find active microangiopathy or inflammation in the resected limbs presumably because of the modification by immune suppressants and complete necrosis.

CT evaluation of the pulmonary lesion at 4 months after the last administration of nivolumab demonstrated significant regression; the tumor had become a scar-like lesion ( Fig. 1b; right panel) and there was no further progression thereafter. The patient was finally discharged from the hospital at 6 months after admission.

Discussion

In this case, the patient had pleiomorphic carcinoma treated with programmed cell death-1 (PD-1) inhibitor (nivolumab) and developed severe CRS. Inhibition of PD-1 mediated immunoregulation dramatically enhances anti-tumor immunity in many cancer patients. On the other hand, 30 to 40% of patients who had received nivolumab for non-small cell carcinoma of the lung developed complication of irAEs [8,9], which often require systemic corticosteroid therapy. It is worth mentioning that patients with irAEs tend to show better progression-free survival in non-small cell lung cancer [10]. This phenomenon suggests that the clinical effects and harmful side-effects of cancer immunotherapy are two sides of the same coin, making cancer immunotherapy that much more difficult. Furthermore, there is no reliable biomarker for estimating clinical efficacy or impending side-effect, although multifarious investigations have been carried out to identify candidate biomarkers from the clinical, pathological, and genetic point of view [8,9]. For example, paraneoplastic syndrome, a relatively rare condition, is considered to be induced by auto-immune reaction to tumor cells [11]. It would follow, therefore, that this syndrome would be a sign of the immunological effect of ICIs; however, there have been no reports showing any correlation between paraneoplastic syndrome and the efficacy of ICIs. Another candidate predictive marker is morphological atypia, which should be associated with tumor mutation burden, being a recently approved indicator of effect of ICIs. Since pleomorphic carcinoma shows significantly high-grade atypia as found in this case, it might be possible to predict both the beneficial and harmful effects of nivolumab. The problem is that quantifying morphological atypia with high reproducibility is challenging in practice. Currently, routine measurement of serum inflammatory cytokines is not common in daily clinical practice. However, monitoring some types of serum cytokines for a certain period would be helpful in predicting the beneficial or detrimental effects of cancer immunotherapy.

We observed increased serum levels of IL-1β, IL-6, TNFα, IFNγ, and G-CSF, which are known to be pleiotropic inflammatory cytokines that produce a myriad of systemic symptoms. In particular, increased IL-6 is a supposed culprit of myocardial disease and coagulopathy, which was observed in this case, although detailed mechanism is still obscure [12]. Clinically identified fever, thrombocytopenia and increased triglyceride, ferritin, and soluble IL-2 receptor levels suggested the presence of HLH, although the widely used Histiocyte Society HLH-2004 diagnostic criteria were not fully met. However, according to the recently proposed HScore evaluation, a novel sophisticated criterion, the score of this patient was 205 corresponding to more than 90% probability of HLH [13]. IL-6 is thought to be involved in the pathogenesis of HLH [14]. In contrast, an interesting report suggested that highly activated CD8 positive T lymphocytes induced regulatory T cell (Treg) dysfunction in patients with HLH [15]. The mechanism is explained by overconsumption of IL-2 by CD8 positive T cells, resulting in decreased Treg number. Indeed, the number of Tregs is significantly decreased in patients with HLH and recovers to normal levels along with clinical improvement. In line with the focus of this report, the basic mechanism of action of ICIs themselves, activation of CD8 positive cells and suppression of    Tregs, can be the fundamental cause of HLH. We suspected the same for secondary TTP as well. We found decreased ADAMTS13 activity, but ADAMTS13 inhibition was not detected. We therefore speculate that the release of multiple inflammatory cytokines hindered the generation of ADAMTS13 from stellate cells, endothelial cells, and platelets, leading to secondary TTP-like microcirculatory impairment resulting in PF, although we cannot exclude a possible subclinical heterogenous ADAMTS13 mutation. This hypothesis is supported by an experimental model where inflammatory cytokines, including TNFα and IFNγ, inhibited synthesis of ADAMTS13 from hepatic stellate cells, a major source of ADAMTS13 [16].

Overall, we propose that all the deleterious hematopoietic adverse effects seen in this case can be attributed to CRS induced by nivolumab-mediated overactivated immune reaction. At the moment, four types of possible mechanisms underlying irAEs have been proposed; overactivation of cellular immune, increasing humoral immune, excessive inflammatory cytokine production and enhancing complement-mediated inflammation [17]. Our current case would be included in "excessive inflammatory cytokine production" pattern. However, we could not identify exact causative factor or specific signaling pathway of this case.

Conclusion

We encountered a case of multiple pathologies including HLH, TTP-like condition, and PF presumably arising from ICI-induced CRS. To our knowledge, ours is the first report to describe this. CRS has been reported in the setting of autoimmune disease and bacterial or viral infection. However, expanding indications and advancing effectiveness of ICIs would increase the incidence of this disease in cancer patients. Although our current case showed significantly high blood concentration of IL-1β, IL-6, TNFα, and IFNγ when compared with previous reports of CRS [18][19][20], we did not use the inhibitory agents for inflammatory cytokines in this case because these were not yet recommended in the actual clinical setting at that time. However, a study reported that administration of anti-IL-6 receptor monoclonal antibody (tocilizumab) was useful for treatment of CRS [12]. Apart from tocilizumab, anti-TNFα and anti-IL-1 receptor monoclonal antibodies are also commercially available. Combination of these anti-cytokine medications would be a promising approach for treating CRS. Accumulating a large number of thoroughly examined cases and the resulting better understanding of this disorder should provide safer cancer immunotherapy.

Table 1
1Laboratory test results on admissionTest
Result Reference Renge
(Unit)

Complete blood
count

Table 1
1Laboratory test results on admission (Continued)Test
Result Reference Renge
(Unit)

IL-10
20.0
< 2.03 (pg/mL)

IL-12
< 0.78 < 3.15 (pg/mL)

TNFα
251
< 1.79 (pg/mL)

INFγ
9260 < 20.6 (pg/mL)

G-CSF
753
10.5-57.5 (pg/mL)

ADAMTS-13
activity

37.8
70.0-120.0 (%)

ADAMTS-13
inhibitor

< 0.5 < 0.5 (BU/mL)

Abbreviations: MCV mean corpuscular volume, MCH mean corpuscular
hemoglobin, MCHC mean corpuscular hemoglobin concentration, PT
prothrombin time, INR international normalized ratio, TAT turnaround time,
AST aspartate aminotransferase, ALT alanine aminotransferase, LDH lactate
dehydrogenase, CK creatine kinase, CK-MB creatine kinase-muscle/brain, CRP
C-reactive protein

AcknowledgementsNot applicable.FundingNone.Availability of data and materials Available upon request.Authors' contributions OH, FS, TN: Involved in care of the patient, wrote and edited this case report. TK, KK, YH: Analyzed clinical and pathological data, wrote and edited this case report. HT, TT conceived of the study and participated in the design, and the preparation of the manuscript. All authors read and approved the final manuscript.Ethics approval and consent to participate Not applicable, not a trial.Consent for publicationAppropriate consent was obtained from the patient.Competing interestsThe authors declare that they have no competing interests.
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