Impact of phytochemicals and plant extracts on viability and proliferation of NK cell line NK-92 - a closer look at immunomodulatory properties of goji berries extract in human colon cancer cells.

INTRODUCTION: Due to the fact that lymphocytes NK (natural killer cells) are the first line of defence of the body against cancer, one of the goals of modern immunotherapy is the enhancement of their natural activities for the effective recognition, detection, and elimination of cancer cells. OBJECTIVE: The aim of the study was to evaluate the influence of selected phytochemicals (curcumin and resveratrol) and plant extracts (chlorella and goji berries) on NK cells viability and proliferation, as well as cytotoxic activity against colon cancer - one of the most common cancer worldwide. MATERIAL AND METHODS: The impact of phytochemicals, viability and proliferation of plant extracts on NK cells was examined in NK-92 cells using both LDH and MTT assays. The immunomodulatory properties of selected compounds were tested against human colon cancer cell line LS180 using the MTT test. RESULTS: Extracts of chlorella and goji berries significantly increased NK cell proliferation, while curcumin and resveratrol did not affect this process. Curcumin, as well as extracts of chlorella and goji berries, did not impact NK viability, while resveratrol significantly increased it. LDH test revealed the cytotoxic effect of chlorella extract and curcumin in NK-92 cell cultures. On the contrary, goji berries extract significantly decreased LDH level, while resveratrol did not affect the integrity of NK cell membranes. Studies conducted in co-cultures NK cells, also directly eliminated colon cancer cells. CONCLUSIONS: Performed studies revealed immunomodulatory properties of goji berries extract, which improved viability and proliferation of NK cells, and above all, significantly increased their ability to recognize and eliminate colon cancer cells.

Induced pluripotent stem cell-derived natural killer cells gene-modified to express chimeric antigen receptor-targeting solid tumors.

The use of allogeneic, pluripotent stem cell-derived immune cells for cancer immunotherapy has been the subject of recent research, including clinical trials. The use of pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, regarding efficacy, safety, and producibility. In this review, we have described the characteristics of natural killer (NK) cells from multiple cell sources, including pluripotent stem cells, the chimeric antigen receptor (CAR)-modification method and strategy for these NK cells, and the current and planned clinical trials of CAR-modified induced pluripotent stem cell-derived NK cells.

Long-chain polyunsaturated omega-3 fatty acids reduce multiple myeloma exosome-mediated suppression of NK cell cytotoxicity.

BACKGROUND: Despite the advances in the treatment of multiple myeloma (MM), complete remission is usually challenging. The interactions between tumor and host cells, in which exosomes (EXs) play critical roles, have been shown to be among the major deteriorative tumor-promoting factors herein. Therefore, any endeavor to beneficially target these EX-mediated interactions could be of high importance. OBJECTIVES: a) To investigate the effects of myeloma EXs on natural killer (NK) cell functions. b) To check whether treatment of myeloma cells with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), two polyunsaturated omega-3 fatty acids with known anti-cancer effects, can modify myeloma EXs in terms of their effects on natural killer functions. METHODS: L363 cells were treated with either EPA or DHA or left untreated and the released EXs (designated as E-EX, D-EX and C-EX, respectively) were used to treat NK cells for functional studies. RESULTS: Myeloma EXs (C-EXs) significantly reduced NK cytotoxicity against K562 cells (P ≤ 0.05), while the cytotoxicity suppression was significantly lower (P ≤ 0.05) in the (E-EX)- and (D-EX)-treated NK cells compared to the (C-EX)-treated cells. The expression of the activating NK receptor NKG2D and NK degranulation, after treatment with the EXs, were both altered following the same pattern. However, C-EXs could increase IFN-γ production in NK cells (P < 0.01), which was not significantly affected by EPA/DHA treatment. This indicates a dual effect of myeloma EXs on NK cells functions. CONCLUSION: Our observations showed that myeloma EXs have both suppressive and stimulatory effects on different NK functions. Treatment of myeloma cells with EPA/DHA can reduce the suppressive effects of myeloma EXs while maintaining their stimulatory effects. These findings, together with the previous findings on the anti-cancer effects of EPA/DHA, provide stronger evidence for the repositioning of the currently existing EPA/DHA supplements to be used in the treatment of MM as an adjuvant treatment. EXs released from L363 (myeloma) cells in their steady state increase IFN-γ production of NK cells, while reduce their cytotoxicity against the K562 cell line (right blue trace). EXs from L363 cells pre-treated with either EPA or DHA are weaker stimulators of IFN-γ production. These EXs also increase NK cytotoxicity and NKG2D expression (left brown trace) compared to the EXs obtained from untreated L363 cells. Based on these findings, myeloma EXs have both suppressive and stimulatory effects on different NK functions depending on the properties of their cells of origin, which can be exploited in the treatment of myeloma.

Concentration of NK cells after ß-glucan and vitamin D supplementation in patients with diabetic retinopathy.

In our study, we focused on possible effects of supplementation with glucan and vitamin D on total numbers of NK cells in patients with diabetic retinopathy. We evaluated possible relations among nutritional state (BMI), leptin levels, and total numbers of NK cells in patients supplemented with (1) glucan and vitamin D, (2) vitamin D and placebo, and (3) vitamin D alone. Our results show that 3 months of supplementation with both glucan and vitamin D resulted in significant improvements of NK cell numbers. In addition, we found statistically significant correlation between NK cell numbers and leptin levels. Based on these results, we propose that the molecule responsible for these changes is glucan, as vitamin D alone or together with placebo caused no effects.

Investigation on activation in RAW264.7 macrophage cells and protection in cyclophosphamide-treated mice of Pseudostellaria heterophylla protein hydrolysate.

Our previous study has demonstrated that Pseudostellaria heterophylla protein hydrolysate (PPH) has immunomodulatory activity on murine spleen lymphocytes. The aim of this study was to investigate the excitation of PPH in RAW264.7 macrophage cells and the protective effect in cyclophosphamide (CTX)-treated mice. The results showed PPH of 50 µg/mL could stimulate macrophages resulting in significant promotions of nitric oxide (NO) production, endocytosis and reactive oxygen species formation. Meanwhile, enzyme-linked immunosorbent assay (ELISA) revealed that the levels of tumor necrosis factor-α and interleukin-10 were significantly upregulated by PPH. Furthermore, 50 mg/kg per day PPH restored the T lymphocyte proliferation and natural killer cell activity, and increased NO production and pinocytosis of peritoneal macrophages in CTX-treated mice. These findings indicate PPH plays a crucial role in RAW264.7 macrophage cells activation and in the protection against immunosuppression in CTX-treated mice and could be used as a potential immunostimulant agent.

The effect of L-theanine supplementation on the immune system of athletes exposed to strenuous physical exercise.

BACKGROUND: The aim of this study was to analyze the response of selected components of the immune system in rowers to maximal physical exercise, and to verify if this response could be modulated by supplementation with L-theanine. METHOD: The double-blind study included 20 members of the Polish Rowing Team. The subjects were randomly assigned to the supplemented group (n = 10), receiving 150 mg of L-theanine extract for 6 weeks, or to the placebo group (n = 10). The participants performed a 2000-m test on a rowing ergometer at the beginning (1st examination) and at the end of the supplementation period (2nd examination). Blood samples were obtained from the antecubital vein before each exercise test, 1 min after completing the test, and after a 24-h recovery. Subpopulations of T regulatory lymphocytes (Tregs) (CD4+/CD25+/CD127-), cytotoxic lymphocytes (CTLs) (CD8+/TCRαß+), natural killer (NK) cells (CD3-/CD16+/CD56+) and TCRδγ-positive (Tδγ) cells were determined by means of flow cytometry. The levels of interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10), interferon gamma (INF-ɤ) and total antioxidant capacity (TAC) were determined with commercially available diagnostic kits. RESULTS: Supplementation with L-theanine contributed to a significant post-exercise decrease in IL-10 concentration, which was reflected by higher values of IL-2 to IL-10 and IFN-γ to IL-10 ratios. Moreover, a significant post-recovery decrease in CTL count, Treg to NK and Treg to CTL ratios was observed in the supplemented group. CONCLUSION: Despite the decrease in the number of some cytotoxic cells (CTLs) and an increase in the proportion of Tregs to CTLs, supplementation with LTE seems to exert a beneficial effect on a disrupted Th1/Th2 balance in elite athletes, as shown by the decrease in IL-10 concentration.

Buyang Huanwu Decoction Attenuates Infiltration of Natural Killer Cells and Protects Against Ischemic Brain Injury.

BACKGROUND/AIMS: Natural killer (NK) cells are among the first immune cells that respond to an ischemic insult in human brains. The infiltrated NK cells damage blood-brain barrier (BBB) and exacerbate brain infarction. Buyang Huanwu Decoction (BHD), a classic Chinese traditional herbal prescription, has long been used for the treatment of ischemic stroke. The present study investigated whether BHD can prevent brain infiltration of NK cells, attenuate BBB disruption and improve ischemic outcomes. METHODS: Transient focal cerebral ischemia was induced in rats by a 60-minute middle cerebral artery occlusion, and BHD was orally administrated at the onset of reperfusion, 12 hours later, then twice daily. Assessed parameters on Day 3 after ischemia were: neurological and motor functional deficits through neurological deficit score and rotarod test, respectively; brain infarction through TTC staining; BBB integrity through Evans blue extravasation; matrix metalloproteinase-2/9 activities through gelatin zymography; tight junction protein, nuclear factor-kB (NF-kB) p65 and phospho-p65 levels through Western blotting; NK cell brain infiltration and CXCR3 levels on NK cells through flow cytometry; interferon-γ production through ELISA; CXCL10 mRNA levels through real-time PCR; CXCL10 expression and p65 nuclear translocation through immunofluorescence staining. RESULTS: BHD markedly reduced brain infarction, improved rotarod performance, and attenuated BBB breakdown. Concurrently, BHD attenuated the upregulation of matrix metalloproteinase-2/9 activities and the degradation of tight junction proteins in the ischemic brain. Infiltration of NK cells was observed in the ischemic hemisphere, and this infiltration was blunted by treatment with BHD. BHD suppressed brain ischemia-induced interferon-γ and chemokine CXCL10 production. Furthermore, BHD significantly reduced the expression of CXCR3 on brain-infiltrated NK cells. Strikingly, BHD did not affect NK cell levels or its CXCR3 expression in the spleen or peripheral blood after brain ischemia. The nuclear translocation of NF-kB p65 and phospho-p65 in the ischemic brain was inhibited by BHD. CONCLUSION: Our findings suggest that BHD prevents brain infiltration of NK cells, preserves BBB integrity and eventually improves ischemic outcomes. The inhibitory effects of BHD on NK cell brain invasion may involve its ability of suppressing NF-kB-associated CXCL10-CXCR3-mediated chemotaxis. Notably, BHD only suppresses NK cells and their CXCR3 expression in the ischemic brain, but not those in periphery.

Psychoneuroimmunology and Natural Killer Cells: The Chromium-Release Whole-Blood Assay.

Natural killer (NK) cells are an essential component of innate immunity. These lymphocytes are also sensitive barometers of the effects of endogenous and exogenous stressors on the immune system. This chapter describes a chromium (51Cr)-release bioassay designed to measure to the target cell killing capacity of NK cells (NKCC). Key features of the cytotoxicity assay are that it is done with whole blood and that numbers of effector cells are determined for each sample by flow cytometry and lymphocyte count. Effector cells are defined as CD3-CD56+ lymphocytes. Target cells are the K562 erythroleukemia cell line. Killing capacity is defined as number of target cells killed per effector cell, at an effector cell/target cell ratio of 1:1 during a 4-h in vitro assay.

Assessment of interactions of efavirenz solid drug nanoparticles with human immunological and haematological systems.

BACKGROUND: Recent work has developed solid drug nanoparticles (SDNs) of efavirenz that have been demonstrated, preclinically, improved oral bioavailability and the potential to enable up to a 50% dose reduction, and is currently being studied in a healthy volunteer clinical trial. Other SDN formulations are being studied for parenteral administration, either as intramuscular long-acting formulations, or for direct administration intravenously. The interaction of nanoparticles with the immunological and haematological systems can be a major barrier to successful translation but has been understudied for SDN formulations. Here we have conducted a preclinical evaluation of efavirenz SDN to assess their potential interaction with these systems. Platelet aggregation and activation, plasma coagulation, haemolysis, complement activation, T cell functionality and phenotype, monocyte derived macrophage functionality, and NK cell function were assessed in primary healthy volunteer samples treated with either aqueous efavirenz or efavirenz SDN. RESULTS: Efavirenz SDNs were shown not to interfere with any of the systems studied in terms of immunostimulation nor immunosuppression. Although efavirenz aqueous solution was shown to cause significant haemolysis ex vivo, efavirenz SDNs did not. No other interaction with haematological systems was observed. Efavirenz SDNs have been demonstrated to be immunologically and haematologically inert in the utilised assays. CONCLUSIONS: Taken collectively, along with the recent observation that lopinavir SDN formulations did not impact immunological responses, these data indicate that this type of nanoformulation does not elicit immunological consequences seen with other types of nanomaterial. The methodologies presented here provide a framework for pre-emptive preclinical characterisation of nanoparticle safety.

An attempt to induce an immunomodulatory effect in rowers with spirulina extract.

BACKGROUND: The aim of this study was to analyze the response of selected components of the immune system in rowers to maximal physical exercise, and to verify if this response can be modulated by supplementation with spirulina (cyanobacterium Spirulina platensis). METHOD: The double-blind study included 19 members of the Polish Rowing Team. The subjects were randomly assigned to the supplemented group (n = 10), receiving 1500 mg of spirulina extract for 6 weeks, or to the placebo group (n = 9). The participants performed a 2000-m test on a rowing ergometer at the beginning (1st examination) and at the end of the supplementation period (2nd examination). Blood samples were obtained from the antecubital vein prior to each exercise test, 1 min after completing the test, and after a 24-h recovery period. Subpopulations of T regulatory lymphocytes (Tregs) [CD4+/CD25+/CD127-], cytotoxic lymphocytes (CTLs) [CD8+/TCRαß+], natural killer (NK) cells [CD3-/CD16+/CD56+] and TCRδγ-positive (Tδγ) cells were determined by means of flow cytometry. RESULTS: On the 2nd examination, athletes from the supplemented group showed neither a post-exercise increase in Treg count nor a post-recovery decrease in Tδγ cell count (both observed in the placebo group), and presented with significantly lower values of Treg/CTL prior to and after the exercise. During the same examination, rowers from the placebo group showed a significant post-recovery increase in Treg/(NK + Tδγ + CTL) ratio, which was absent in the supplemented group. CONCLUSION: The results of this study imply that supplementation with spirulina extract may protect athletes against a deficit in immune function (especially, anti-infectious function) associated with strenuous exercise, and may cause a beneficial shift in "overtraining threshold" preventing a radical deterioration of immunity.

Olive Leaf Extracts Act as Modulators of the Human Immune Response.

BACKGROUND: Olive tree leaves have been used in the Mediterranean area as traditional medicine in virtue of their healthy effects. Olive leaf extracts (OLEs) contain higher amounts of polyphenols than those detected in the extra virgin olive oil and fruit. Several lines of evidence support the cardioprotective, anti-oxidant and anti-inflammatory activities exerted by OLEs. METHODS: Peripheral blood mononuclear cells from twenty-five healthy donors were cultured in the presence of 3 µg of two OLE extracts, extract A (resuspended in water) and extract B (resuspended in 70% ethanol). After harvesting, cell pellets were used for cytofluorimetric phenotyping, while supernatants were assayed for cytokine release by means of ELISA. Furthermore, in the same supernatants nitric oxide (NO) content was determined. RESULTS: Both extracts, but especially extract A, increased absolute numbers of CD8+ and natural killer (NK) cells. In addition, an increased production of interferon (IFN)-γ by both extracts as an expression of T helper (h)1 activation was observed. Finally, both extracts enhanced NO release. CONCLUSION: OLEs, and mostly extract A, are able to in vitro modify healthy human immune response by increasing IFN-γ production which seems to be associated to the higher absolute numbers of CD8+ and NK cells and this may suggest a reinforcement of the anti-tumor activity. Furthermore, increased levels of NO may indicate the potential cardioprotective effects exerted by OLEs in virtue of their vasodilation dependent activity. Finally, OLEs are able to maintain the equilibrium between T regulatory cells and Th17 cells as evidenced by unmodified levels of interleukin (IL)-IL-10 and IL-17, respectively. In the light of these results, OLEs are potential therapeutic compounds for the treatment of chronic inflammatory disease, also preventing cardiovascular event outcome.

Regulation of NKG2D-Dependent NK Cell Functions: The Yin and the Yang of Receptor Endocytosis.

Natural-killer receptor group 2, member D (NKG2D) is a well characterized natural killer (NK) cell activating receptor that recognizes several ligands poorly expressed on healthy cells but up-regulated upon stressing stimuli in the context of cancer or viral infection. Although NKG2D ligands represent danger signals that render target cells more susceptible to NK cell lysis, accumulating evidence demonstrates that persistent exposure to ligand-expressing cells causes the decrease of NKG2D surface expression leading to a functional impairment of NKG2D-dependent NK cell functions. Upon ligand binding, NKG2D is internalized from the plasma membrane and sorted to lysosomes for degradation. However, receptor endocytosis is not only a mechanism of receptor clearance from the cell surface, but is also required for the proper activation of signalling events leading to the functional program of NK cells. This review is aimed at providing a summary of current literature relevant to the molecular mechanisms leading to NKG2D down-modulation with particular emphasis given to the role of NKG2D endocytosis in both receptor degradation and signal propagation. Examples of chronic ligand-induced down-regulation of NK cell activating receptors other than NKG2D, including natural cytotoxicity receptors (NCRs), DNAX accessory molecule-1 (DNAM1) and CD16, will be also discussed.

Engineering Natural Killer Cells for Cancer Immunotherapy.

The past several years have seen tremendous advances in the engineering of immune effector cells as therapy for cancer. While chimeric antigen receptors (CARs) have been used extensively to redirect the specificity of autologous T cells against hematological malignancies with striking clinical results, studies of CAR-modified natural killer (NK) cells have been largely preclinical. In this review, we focus on recent advances in NK cell engineering, particularly on preclinical evidence suggesting that NK cells may be as effective as T cells in recognizing and killing targets after genetic modification. We will discuss strategies to introduce CARs into both primary NK cells and NK cell lines in an effort to provide antigen specificity, the challenges of manufacturing engineered NK cells, and evidence supporting the effectiveness of this approach from preclinical and early-phase clinical studies using CAR-engineered NK cells. CAR-NK cells hold great promise as a novel cellular immunotherapy against refractory malignancies. Notably, NK cells can provide an "off-the-shelf" product, eliminating the need for a personalized and patient-specific product that plagues current CAR-T cell therapies. The ability to more potently direct NK cell-mediated cytotoxicity against refractory tumors through the expression of CAR is likely to contribute to the recent paradigm shift in cancer treatment.

Immunomodulatory and cellular anti-oxidant activities of caffeic, ferulic, and p-coumaric phenolic acids: a structure-activity relationship study.

Many studies have been performed to assess the potential utility of natural products as immunomodulatory agents to enhance host responses and to reduce damage to the human body. To determine whether phenolic compounds (caffeic, ferulic, and p-coumaric acids) have immunomodulatory effects and clarify which types of immune effector cells are stimulated in vitro, we evaluated their effect on splenocyte proliferation and lysosomal enzyme activity. We also investigated the activity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL). In addition, induction of the cellular antioxidant activity in splenocytes, macrophages, and red blood cells was determined by measuring the fluorescence of the DCF product. The study first results indicated that caffeic, ferulic, and p-coumaric acids significantly promote LPS-stimulated splenocyte proliferation, suggesting a potential activation of B cells, and enhanced humoral immune response in hosts treated by the tested natural products. Phenolic acids significantly enhanced the killing activity of isolated NK and CTL cells but had negligible effects on mitogen-induced proliferation of splenic T cells. We showed that caffeic acid enhances lysosomal enzyme activity in murine peritoneal macrophages, suggesting a potential role in activating such cells. Immunomodulatory activity was concomitant with the cellular antioxidant effect in macrophages and splenocytes of caffeic and ferulic acids. We conclude from this study that caffeic, ferulic, and p-coumaric acids exhibited an immunomodulatory effect which could be ascribed, in part, to their cytoprotective effect via their antioxidant capacity. Furthermore, these results suggest that these natural products could be potentially used to modulate immune cell functions in physiological and pathological conditions.

Intralipid supplementation in women with recurrent spontaneous abortion and elevated levels of natural killer cells.

OBJECTIVE: To investigate the efficacy of intralipid supplementation in women with recurrent spontaneous abortion (RSA) and elevated natural killer cell activity undergoing in vitro fertilization/intracytoplasmic sperm injection. METHODS: Between February 10, 2013, and April 30, 2015, a double-blind randomized controlled study was conducted at a center in Egypt. Women with unexplained secondary infertility, RSA, and elevated levels of natural killer cells (>12%) were enrolled and randomly assigned to receive intralipid (2mL diluted at 20% in 250mL saline) or saline (250mL) infusion on the day of oocyte retrieval using random numbers and sealed envelopes. Patients and attending physicians were masked to group assignment. The infusions were repeated within 1week of a positive pregnancy test and then every 2weeks until the end of the first trimester. The primary outcome was chemical pregnancy 14days after embryo transfer. Analyses were by intention-to-treat. RESULTS: Overall, 296 women were enrolled. Chemical pregnancy was recorded for 84 (58.3%) of 144 women in the intralipid group and 76 (50.0%) of 152 in the control group (P=0.129). CONCLUSION: Intralipid supplementation did not increase frequency of chemical pregnancy. However, findings related to ongoing pregnancy and live birth should be investigated further. ClinicalTrials.gov:NCT01788540.

Zinc and immunity: An essential interrelation.

The significance of the essential trace element zinc for immune function has been known for several decades. Zinc deficiency affects immune cells, resulting in altered host defense, increased risk of inflammation, and even death. The micronutrient zinc is important for maintenance and development of immune cells of both the innate and adaptive immune system. A disrupted zinc homeostasis affects these cells, leading to impaired formation, activation, and maturation of lymphocytes, disturbed intercellular communication via cytokines, and weakened innate host defense via phagocytosis and oxidative burst. This review outlines the connection between zinc and immunity by giving a survey on the major roles of zinc in immune cell function, and their potential consequences in vivo.

Innate lymphoid cells: parallel checkpoints and coordinate interactions with T cells.

Protection of epithelial and mucosal surfaces is required for survival. The recent discovery of a diverse array of innate lymphoid cells that lie immediately beneath these surfaces has unexpectedly uncovered an entire defense system distinct from the adaptive system essential to protect these barriers. This multilayered design provides a robust system through coupling of two highly complementary networks to ensure immune protection. Here, we discuss the similarities in the hardwiring and diversification of innate lymphoid cells and T cells during mammalian immune responses.

Anticancer and immunostimulating activities of a novel homogalacturonan from Hippophae rhamnoides L. berry.

Our previous study isolated an anti-fatigue polysaccharide (HRWP) from the Hippophae rhamnoides berry. In this study, using ion-exchange chromatography and gel filtration chromatography in turn, a water-soluble homogenous polysaccharide HRWP-A was isolated from HRWP. Structural analysis determined that HRWP-A was a polysaccharide with repeating units of (1→4)-ß-d-galactopyranosyluronic residues, of which 85.16% were esterified with methyl groups. An antitumor activity assay showed that HRWP-A could significantly inhibit the Lewis lung carcinoma (LLC) growth in tumor-bearing mice. Further experiments suggested that the antitumor effect of HRWP-A might be mediated through immunostimulating activity, as it enhances the lymphocyte proliferation, augments the macrophage activities, as well as promoting NK cell activity and CTL cytotoxicity in tumor-bearing mice. To our knowledge, this is the first report on a natural antitumor high-methoxyl homogalacturonan pectin from the H. rhamnoides berry-a compound that acts as a potential immunostimulant and anticancer adjuvant.

Comparison of physicochemical properties and immunomodulatory activity of polysaccharides from fresh and dried litchi pulp.

Drying is commonly used for preservation and processing of litchi. However, its polysaccharide structure may be altered by the drying process, resulting in biological activity changes. Polysaccharides from fresh and dried litchi pulp (denoted as LPF and LPD, respectively) were isolated, investigated by GC-MS, GPC and UV/IR spectrum analysis and their antitumor and immunomodulatory activities were evaluated in vitro. LPD, the molecular weight of which was lower than that of LPF, contained more protein, uronic acid, arabinose, galactose and xylose. Compared with LPF, LPD exhibited a higher inhibitory effect on the proliferation of HepG2, Hela and A549 cells from 50-750 µg/mL. LPD was also a better stimulator of spleen lymphocyte proliferation, NK cells cytotoxicity and macrophage phagocytosis from 50-400 µg/mL. In summary, drying could change the physicochemical properties and enhance the bioactivity of polysaccharides from litchi pulp. This finding is supported by the fact that dried litchi pulps are used in Traditional Chinese Medicine.

Vitamin D deficiency may be a risk factor for recurrent pregnancy losses by increasing cellular immunity and autoimmunity.

STUDY QUESTION: Do women with recurrent pregnancy losses (RPL) and low vitamin D have increased prevalence of auto- and cellular immune abnormalities when compared with women with RPL who have normal vitamin D, and does vitamin D have any effect on cellular immunity in vitro? SUMMARY ANSWER: A high proportion of women with RPL have vitamin D deficiency and the risk of auto- and cellular immune abnormalities is increased in women with RPL and vitamin D deficiency. WHAT IS KNOWN ALREADY: Vitamin D deficiency in pregnant women is associated with increased risk of obstetrical complications such as pre-eclampsia, bacterial vaginosis associated preterm delivery, gestational diabetes mellitus and small-for-gestational age births. STUDY DESIGN, SIZE, DURATION: A retrospective cross-sectional study of 133 women with RPL who were enrolled in a 2-year period, together with laboratory experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women with three or more consecutive spontaneous abortions prior to 20 weeks of gestation who were enrolled at the University clinic. Serum vitamin D level, cellular activity and autoimmune parameters in vivo and in vitro were measured. MAIN RESULTS AND THE ROLE OF CHANCE: Sixty-three out of 133 women (47.4%) had low vitamin D (<30 ng/ml). The prevalence of antiphospholipid antibody (APA) was significantly higher in low vitamin D group (VDlow) (39.7%) than in the normal vitamin D group (VDnl) (22.9%) (P< 0.05) and the adjusted odds ratio (OR) for APA in VDlow was 2.22 with the 95% confidence interval (CI) of 1.0-4.7. The prevalence of antinuclear antigen antibody (VDlow versus VDnl; 23.8% versus 10.0%, OR 2.81, 95% CI 1.1-7.4), anti-ssDNA (19.0% versus 5.7%, OR 3.76, 95% CI 1.1-12.4) and thyroperoxidase antibody (33.3% versus 15.7%, OR 2.68, 95% CI 1.2-6.1) was significantly higher in VDlow than those of VDnl (P < 0.05 each). Peripheral blood CD19(+) B and CD56(+) NK cell levels and NK cytotoxicity at effector to target cell (E:T) ratio of 25:1 were significantly higher in VDlow when compared with those of VDnl (P < 0.05 each). Reduction (%) of NK cytotoxicity (at E:T ratio of 50:1 and 25:1) by IgG (12.5 mg/dl) was significantly lower in VDlow than those of VDnl (P < 0.05, P < 0.01, respectively). There were no differences in Th1/Th2 ratios between VDlow and VDnl. When vitamin D3 was added in NK cytotoxicity assay in vitro, NK cytotoxicity at E:T ratio of 50:1 was significantly suppressed with 10 nMol/L (nM) (11.9 ± 3.3%) and 100 nM (10.9 ± 3.7%) of vitamin D3 when compared with controls (15.3 ± 4.7%) (P < 0.01 each). TNF-α/IL-10 expressing CD3(+)/4(+) cell ratios were significantly decreased with 100 nM of vitamin D3 (31.3 ± 9.4, P < 0.05) when compared with controls (40.4 ± 11.3) in vitro. Additionally, INF-γ/IL-10 expressing CD3(+)/4(+) cell ratio was significantly decreased with 100 nM of vitamin D3 (12.1 ± 4.0, P < 0.05) when compared with controls (14.8 ± 4.6). IFN-γ and TNF-α secretion from NK cells were significantly decreased (P < 0.01 each), and IL-10, IL-1ß, vascular endothelial growth factor and granulocyte colony stimulating factor levels were significantly increased (P < 0.01 each) with vitamin D3 100 nM when compared with those of controls. LIMITATIONS, REASONS FOR CAUTION: The prevalence of vitamin D deficiency in women with RPL in this study is open to a possible type I error since women with vitamin D supplementation were excluded from this study. WIDER IMPLICATIONS OF THE FINDINGS: Assessment of vitamin D level is recommended in women with RPL. Vitamin D supplementation should be explored further as a possible therapeutic option for RPL. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the intramural funding from Department of Microbiology and Immunology, Chicago Medical School at Rosalind Franklin University of Medicine and Science. None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.