Decrease of Peripheral and Intestinal NKG2A-Positive T Cells in Patients with Ulcerative Colitis

To investigate the role of inhibitory natural killer receptors (iNKRs) in inflammatory bowel disease (IBD), we analyzed the expression of NKG2A, one of the iNKRs, on T cells in a mouse colitis model and human IBD. During the active phase of dextran sulfate sodium (DSS)-induced mouse colitis, the frequency of NKG2A+ T cells was significantly decreased in the peripheral blood, and increased in the intestine, suggesting the mobilization of this T cell subset to the sites of inflammation. Administration of anti-NKG2A antibody increased the number of inflammatory foci in DSS-induced colitis, suggesting the involvement of NKG2A+ T cells in this colitis model. In ulcerative colitis (UC) patients, the frequency of peripheral blood NKG2A+ T cells was significantly decreased, compared with Crohn's disease (CD) patients and healthy controls, regardless of clinical conditions such as treatment modalities and disease activity. Notably, in sharp contrast to the DSS-induced mouse colitis model, the frequency of NKG2A+ cells among intestinal T cells was also decreased in UC patients. These results suggest that inadequate local infiltration of NKG2A+ T cells may be involved in the pathogenesis of UC.     

非霍奇金淋巴瘤患者外周血中CD4+CD25+ 调节性T 细胞 亚群变化初探

目的:检测非霍奇金淋巴瘤(non-Hodgkin’s lymphoma,NHL)患者外周血中CD4+CD25+调节性T细胞(CD4+CD25+regulatoryT cell,Treg)的改变,探讨Treg与NHL的相关性。方法:病例组(n=60)为本院收治的初诊NHL患者,对照组(n=60)为本院健康体检者,用流式细胞技术联合标记CD4、CD25检测对照组及病例组化疗前、化疗后的外周血中CD4+CD25+调节性T细胞的分布特点。结果:(1)病例组化疗前外周血中CD4+细胞比例显著低于对照组(P<0.05),CD4+CD25+调节性T细胞比例显著高于对照组(P<0.05);(2)病例组化疗后,CD4+细胞比例明显高于化疗前(P<0.05),CD4+CD25+调节性T细胞比例明显低于化疗前(P<0.05);(3)病例组化疗后CD4+细胞比例与对照组无显著差异(P>0.05),而CD4+CD25+调节性T细胞比例显著高于对照组(P<0.05)。结论:非霍奇金淋巴瘤患者外周血中CD4+CD25+调节性T细胞比例升高,存在机体免疫抑制,化疗可降低CD4+CD25+调节性T细胞比例。     

SLE带状疱疹患者外周血CD4~+CD28~+和CD4~+CD25~+FoxP3~+调节性T细胞的表达及相关性研究

目的:检测系统性红斑狼疮(systemic lupus erythematosus,SLE)合并带状疱疹患者外周血CD4~+CD28~+和CD4~+CD25~+Fox P3~+调节性T细胞的表达及相关性,探讨其在SLE合并带状疱疹发病中的临床意义。方法:采用流式细胞术检测30例SLE患者、30例SLE合并带状疱疹患者及30例健康对照者外周血中CD4~+/CD8~+T淋巴细胞亚群表面CD28的表达及CD4~+CD25~+Fox P3~+Treg细胞的表达水平,并分析SLE合并带状疱疹患者外周血CD4~+CD28~+和CD4~+CD25~+Fox P3~+调节性T细胞表达的相关性。结果:SLE合并带状疱疹组患者急性期外周血CD4~+T淋巴细胞比率、绝对计数显著降低,CD4~+、CD8~+T淋巴细胞表面的CD28表达下调,CD4~+CD25~+Fox P3~+Treg细胞水平显著高于SLE组及健康对照组,SLE合并带状疱疹组患者外周血CD4~+CD25~+Fox P3~+Treg水平与CD4~+CD28~+水平成负相关(P均0.05)。结论:SLE合并带状疱疹患者CD4~+、CD8~+T细胞活化异常,CD4~+CD25~+Fox P3~+Treg细胞可能参与抑制了T细胞的活化。     

Low Frequency of Circulating CD8+ T Stem Cell Memory Cells in Chronic Chagasic Patients with Severe Forms of the Disease

Background

CD8+ T cells have been shown to play a crucial role in Trypanosoma cruzi infection. Memory CD8+ T cells can be categorised based on their distinct differentiation stages and functional activities as follows: stem cell memory (TSCM), central memory (TCM), transitional memory (TTM), effector memory (TEM) and terminal effector (TTE) cells. Currently, the immune mechanisms that control T. cruzi in the chronic phase of the infection are unknown.

Methodology/Principal Findings

To characterise the CD8+ T cell subsets that could be participating in the control of T. cruzi infection, in this study, we compared total and T. cruzi-specific circulating CD8+ T cells with distinctive phenotypic and functional features in chronic chagasic patients (CCPs) with different degrees of cardiac dysfunction. We observed a decreased frequency of total TSCM along with an increased frequency of TTE in CCPs with severe disease. Antigen-specific TSCM cells were not detectable in CCPs with severe forms of the disease. A functional profile of CD8+ T cell subsets among CCPs revealed a high frequency of monofunctional CD8+ T cells in the most severe patients with IFN-γ+- or TNF-α+-producing cells.

Conclusions/Significance

These findings suggest that CD8+ TSCM cells may be associated with the immune response to T. cruzi and outcome of Chagas disease, given that these cells may be involved in repopulating the T cell pool that controls infection.     

狼疮性肾炎患者血清NETs、TWEAK、外周血CD4+T/CD8+T比例与疾病活动度及肾脏预后的关系

摘要 目的：探讨狼疮性肾炎（LN）患者血清中性粒细胞胞外诱捕网（NETs）、肿瘤坏死因子样凋亡微弱诱导剂（TWEAK）、外周血分化簇（CD）4⁺T/CD8⁺T比例与疾病活动度及肾脏预后的关系。方法：选取2021年8月～2022年8月川北医学院附属医院肾内科收治的LN患者137例（LN组）,根据系统性红斑狼疮疾病活动指数（SLEDAI）-2000评分分为轻度活动组（52例）、中度活动组（45例）、重度活动组（40例）。随访1年,根据肾脏相关终点事件发生情况分为预后不良组（43例）和预后良好组（94例）,另选取同期76名体检健康志愿者（对照组）。采用酶联免疫吸附法检测血清NETs、TWEAK水平,流式细胞术检测外周血CD4⁺T/CD8⁺T比例。Spearman相关性分析LN患者血清NETs、TWEAK和外周血CD4⁺T/CD8⁺T与SLEDAI-2000评分的相关性,多因素Logistic回归分析LN患者预后不良的因素,受试者工作特征曲线分析血清NETs、TWEAK和外周血CD4⁺T/CD8⁺T对LN患者预后不良的预测价值。结果：与对照组比较,LN组血清NETs、TWEAK水平升高,外周血CD4⁺T/CD8⁺T降低（P＜0.05）。轻度活动组、中度活动组、重度活动组血清NETs、TWEAK依次升高,外周血CD4⁺T/CD8⁺T依次降低（P＜0.05）。LN患者SLEDAI-2000评分与血清NETs、TWEAK呈正相关,与外周血CD4⁺T/CD8⁺T呈负相关（P＜0.05）。慢性肾脏病分期4期、SLEDAI-2000评分升高、NETs升高、TWEAK升高为LN患者预后不良的独立危险因素,估算肾小球滤过率升高、CD4⁺T/CD8⁺T升高为独立保护因素（P＜0.05）。血清NETs、TWEAK和外周血CD4⁺T/CD8⁺T联合预测LN患者预后不良的曲线下面积为0.943,大于血清NETs、TWEAK和外周血CD4⁺T/CD8⁺T单独预测的0.790、0.788、0.799（P＜0.05）。结论：LN患者血清NETs、TWEAK水平升高,外周血CD4⁺T/CD8⁺T降低,与疾病活动度及肾脏预后不良密切相关,血清NETs、TWEAK联合外周血CD4⁺T/CD8⁺T预测LN患者肾脏预后的价值较高。     

原发性肝癌患者Th1/Th2 亚群研究*

目的:探讨原发性肝癌患者血清中淋巴细胞亚群中Th1/Th2的变化,为分析肝癌的发生发展状症和临床治疗提供免疫学指标。方法:应用放射免疫分析及酶联免疫分析法(ELISA),测定46例肝癌患者,及43例正常对照组进行比较。以IL-2、INF-γ和TNF-α水平代表Th1型细胞因子,以IL-4,IL-6、IL-8、IL-10的水平代表Th2型细胞因子。结果:肝癌患者IL-2、TNF-γ、IL-6的水平明显低正常对照组,P<0.01。IL-4、IL-8、IL-10、TNF-α的水平明显高于正常对照组,P<0.01。结论:肝癌患者体内存在Th1/Th2细胞因子失衡,其中Th1亚群功能抑制,Th2亚群功能亢进,其与肿瘤在宿主体内生长密切相关。通过纠正这些免疫失调将成为肝癌治疗的重要手段。     

Decreased Expression of miR-21, miR-26a,miR-29a,and miR-142-3p in CD4+ T Cells and Peripheral Blood from Tuberculosis Patients

The vast majority of Mycobacterium tuberculosis (M. tuberculosis) infected individuals are protected from developing tuberculosis and T cells are centrally involved in this process. MicroRNAs (miRNA) regulate T-cell functions and are biomarker candidates of disease susceptibility and treatment efficacy in M. tuberculosis infection. We determined the expression profile of 29 selected miRNAs in CD4⁺ T cells from tuberculosis patients and contacts with latent M. tuberculosis infection (LTBI). These analyses showed lower expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4⁺ T cells from tuberculosis patients. Whole blood miRNA candidate analyses verified decreased expression of miR-26a, miR-29a, and miR-142-3p in children with tuberculosis as compared to healthy children with LTBI. Despite marked variances between individual donor samples, trends of increased miRNA candidate expression during treatment and recovery were observed. Functional in vitro analysis identified increased miR-21 and decreased miR-26a expression after re-stimulation of T cells. In vitro polarized Interleukin-17 positive T-cell clones showed activation-dependent miR-29a up-regulation. In order to characterize the role of miR-29a (a described suppressor of Interferon-γ in tuberculosis), we analyzed M. tuberculosis specific Interferon-γ expressing T cells in children with tuberculosis and healthy contacts but detected no correlation between miR-29a and Interferon-γ expression. Suppression of miR-29a in primary human T cells by antagomirs indicated no effect on Interferon-γ expression after in vitro activation. Finally, classification of miRNA targets revealed only a moderate overlap between the candidates. This may reflect differential roles of miR-21, miR-26a, miR-29a, and miR-142-3p in T-cell immunity against M. tuberculosis infection and disease.     

Patients with Tuberculosis Disease Have Mycobacterium tuberculosis-Specific CD8 T Cells with a Pro-Apoptotic Phenotype and Impaired Proliferative Capacity,Which Is Not Restored following Treatment

CD8 T cells play a critical role in control of chronic viral infections; however, the role of these cells in containing persistent bacterial infections, such as those caused by Mycobacterium tuberculosis (Mtb), is less clear. We assessed the phenotype and functional capacity of CD8 T cells specific for the immunodominant Mtb antigens CFP-10 and ESAT-6, in patients with pulmonary tuberculosis (TB) disease, before and after treatment, and in healthy persons with latent Mtb infection (LTBI). In patients with TB disease, CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells had an activated, pro-apoptotic phenotype, with lower Bcl-2 and CD127 expression, and higher Ki67, CD57, and CD95 expression, than in LTBI. When CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells were detectable, expression of distinct combinations of these markers was highly sensitive and specific for differentiating TB disease from LTBI. Successful treatment of disease resulted in changes of these markers, but not in restoration of CFP-10/ESAT-6-specific CD8 or CD4 memory T cell proliferative capacity. These data suggest that high mycobacterial load in active TB disease is associated with activated, short-lived CFP-10/ESAT-6-specific CD8 T cells with impaired functional capacity that is not restored following treatment. By contrast, LTBI is associated with preservation of long-lived CFP-10/ESAT-6-specific memory CD8 T cells that maintain high Bcl-2 expression and which may readily proliferate.     

健脾消瘿汤联合优甲乐对脾气亏虚型桥本甲状腺炎患者甲状腺激素、甲状腺自身抗体和CD4+CD45RO+记忆性T细胞的影响

摘要 目的：探讨健脾消瘿汤联合优甲乐对脾气亏虚型桥本甲状腺炎（HT）患者甲状腺激素、甲状腺自身抗体和CD4+CD45RO+记忆性T细胞的影响。方法：选择2020年5月~2021年11月期间我院收治的HT患者60例，采用随机数字表法分为对照组（优甲乐治疗，30例）和研究组（健脾消瘿汤联合优甲乐治疗，30例），两组均治疗12周后观察临床疗效，并对比两组治疗前后中医证候积分、甲状腺激素、甲状腺自身抗体和CD4+CD45RO+记忆性T细胞的变化。结果：研究组临床总有效率较对照组高（P<0.05）。研究组治疗后促甲状腺激素（TSH）水平、中医证候总积分、CD4+CD45RO+记忆性T细胞在外周血单个核细胞（PBMC）中的百分比、甲状腺球蛋白抗体（TGAb）、甲状腺过氧化物酶抗体（TPOAb）均低于对照组（P<0.05），游离三碘甲状腺原氨酸（FT3）、游离甲状腺素（FT4）水平高于对照组（P<0.05）。结论：脾气亏虚型HT患者采用健脾消瘿汤联合优甲乐治疗，可改善其甲状腺激素水平，调节甲状腺自身抗体水平和CD4+CD45RO+记忆性T细胞在PBMC中的百分比，进一步提高临床疗效。     

Processing, secretion, and anti-HIV-1 activity of IL-16 with or without a signal peptide in CD4+ T cells

CD4+ T cells transfected with the C-terminal 130 aa of human IL-16 are rendered resistant to HIV infection. Whether the constitutively expressed IL-16 acts intracellularly, extracellularly, or both is not clear. To address this question and to further study the processing of IL-16, new constructs containing either the C-terminal 130 aa or the C-terminal 100 aa (PDZ-like motif) were constructed with and without a signal peptide. Pulse-chase experiments and treatment of cells with brefeldin A and/or tunicamycin showed that IL-16 is secreted despite the absence of a signal peptide, but with a signal peptide IL-16 is processed through the endoplasmic reticulum-golgi pathway and is glycosylated. Cells expressing IL-16 linked to a signal peptide secrete considerably more IL-16 into the supernatant than cells expressing IL-16 without a signal peptide and are considerably more resistant to HIV replication. Resistance extends to almost 25 days for cells expressing IL-16 with signal peptide as compared with only 15 days for cells without signal peptide. Cells expressing the C-terminal 100 aa not linked to a signal peptide are poor secretors of IL-16 and show little if any resistance to HIV. In contrast, cells expressing the C-terminal 100 aa linked to a signal peptide secrete IL-16 and are resistant to HIV replication. It is concluded that the secretion of IL-16 is required for HIV inhibition.     

Dermal Mesenchymal Stem Cells (DMSCs) Inhibit Skin-Homing CD8+ T Cell Activity,a Determining Factor of Vitiligo Patients’ Autologous Melanocytes Transplantation Efficiency

We here investigated the efficiency of autologous melanocyte transplantation of 23 vitiligo patients by focusing on perilesional skin homing CD8+ T lymphocytes, and studied the potential effect of dermal mesenchymal stem cells (DMSCs) on CD8+ T cell activities in vitro. Out of 23 patients with the autologous melanocyte transplantation, 12 patients (52.17%) had an excellent re-pigmentation, 6 patients (26.09%) had a good re-pigmentation, 5 patients (21.74%) had a fair or poor re-pigmentation. CD8+ T cells infiltrating was observed in the perilesional vitiligo area of all patients. Importantly, the efficiency of the transplantation was closely associated with skin-homing CD8+ T cell activities. The patients with high number of perilesional CD8+ T cells or high level of cytokines/chemokines were associated with poor re-pigmentation efficiency. For in-vitro experiments, we successfully isolated and characterized human DMSCs and skin-homing CD8+ T cells. We established DMSCs and CD8+ T cell co-culture system, where DMSCs possessed significant inhibitory effects against skin homing CD8+ T lymphocytes. DMSCs inhibited CD8+ T cells proliferation, induced them apoptosis and regulated their cytokines/chemokines production. Our results suggest that vitiligo patients’ autologous melanocytes transplantation efficiency might be predicted by perilesional skin-homing CD8+ T cell activities, and DMSCs might be used as auxiliary agent to improve transplantation efficacy.     

Intestinal Parasitosis in Relation to CD4+T Cells Levels and Anemia among HAART Initiated and HAART Naive Pediatric HIV Patients in a Model ART Center in Addis Ababa,Ethiopia

BackgroundIntestinal parasites (IPs) are major concerns in most developing countries where HIV/AIDS cases are concentrated and almost 80% of AIDS patients die of AIDS-related infections. In the absence of highly active antiretroviral therapy (HAART), HIV/AIDS patients in developing countries unfortunately continue to suffer from the consequences of opportunistic and other intestinal parasites. The aim of the study was to determine the prevalence of intestinal parasites in relation to CD4+ T cells levels and anemia among HAART initiated and HAART naïve pediatric HIV patients in a Model ART center in Addis Ababa, Ethiopia.MethodsA prospective comparative cross-sectional study was conducted among HAART initiated and HAART naive pediatric HIV/AIDS patients attending a model ART center at Zewditu Memorial Hospital between August 05, 2013 and November 25, 2013. A total of 180 (79 HAART initiated and 101 HAART naïve) children were included by using consecutive sampling. Stool specimen was collected and processed using direct wet mount, formol-ether concentration and modified Ziehl-Neelsen staining techniques. A structured questionnaire was used to collect data on socio-demographic and associated risk factors. CD4+ T cells and complete blood counts were performed using BD FACScalibur and Cell-Dyn 1800, respectively. The data was analyzed by SPSS version 16 software. Logistic regressions were applied to assess any association between explanatory factors and outcome variables. P values < 0.05 were taken as statistically significant.ResultsThe overall prevalence of IPs was 37.8% where 27.8% of HAART initiated and 45.5% of HAART naive pediatric HIV/AIDS patients were infected (p < 0.05). Cryptosporidium species, E. histolytica/dispar, Hook worm and Taenia species were IPs associated with CD4+ T cell counts <350 cells/μμL in HAART naive patients. The overall prevalence of anemia was 10% in HAART and 31.7% in non-HAART groups. Hook worm, S. stercoralis and H. nana were helminthes significantly associated with anemia in non-HAART patients [AOR, 95% CI: 4.5(1.3, 15.2), P< 0.05]. The prevalence of IPs in non-HAART patients was significantly associated with eating unwashed/raw fruit [AOR, 95%CI: 6.3(1.2, 25.6), P<0.05], open field defecation [AOR, 95%CI: 9.3(1.6, 53.6), P<0.05] and diarrhea [AOR, 95%CI: 5.2(1.3, 21.3), P<0.05]. IPs significantly increased in rural residents [AOR, 95%CI: 0.4(0.1, 0.9, P<0.05)].ConclusionThe overall prevalence of intestinal parasites significantly differed by HAART status and cryptosporidium species were found only in HAART naïve patients with low CD4+ T cell counts. Anemia was also more prevalent and significantly associated with IPs in non-HAART patients. This study identified some environmental and associated risk factors for intestinal parasitic infections. Therefore, Public health measures should continue to emphasize the importance of environmental and personal hygiene to protect HIV/AIDS patients from infections with intestinal parasites and maximize the benefits of HAART.     

One Dose of Staphylococcus aureus 4C-Staph Vaccine Formulated with a Novel TLR7-Dependent Adjuvant Rapidly Protects Mice through Antibodies,Effector CD4+ T Cells,and IL-17A

A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4⁺ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4⁺ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.     

五灵胶囊联合富马酸丙酚替诺福韦片对慢性乙型肝炎患者氧化应激、CD4+CD25+调节性T细胞和血清MMP-1、MMP-2、TIMP-1的影响

摘要 目的：探讨五灵胶囊联合富马酸丙酚替诺福韦片对慢性乙型肝炎（CHB）患者氧化应激、CD4⁺CD25⁺调节性T细胞和血清基质金属蛋白酶-1（MMP-1）、基质金属蛋白酶-2（MMP-2）、基质金属蛋白酶组织抑制因子-1（TIMP-1）的影响。方法：纳入苏州大学附属传染病医院2021年6月~2022年12月期间收治的CHB患者122例,采用随机数字表法分为对照组（n=61,富马酸丙酚替诺福韦片）和研究组（n=61,五灵胶囊联合富马酸丙酚替诺福韦片）。对比两组疗效、氧化应激指标、CD4⁺CD25⁺调节性T细胞、肝功能指标[总胆红素（TBIL）、谷丙转氨酶（ALT）、谷氨酰转肽酶（GGT）]、血清MMP-1、MMP-2、TIMP-1水平和不良反应发生情况。结果：研究组的临床总有效率高于对照组（P<0.05）。两组治疗后丙二醛（MDA）下降,且研究组低于对照组同时间点;超氧化物歧化酶（SOD）升高,且研究组高于对照组同时间点（P<0.05）。两组治疗后CD4⁺CD25⁺调节性T细胞下降,且研究组低于对照组同时间点（P<0.05）。两组治疗后MMP-1、MMP-2、TIMP-1下降,且研究组低于对照组同时间点（P<0.05）。两组治疗后TBIL、ALT、GGT下降,且研究组低于对照组同时间点（P<0.05）。两组不良反应总发生率组间对比未见差异（P>0.05）。结论：五灵胶囊联合富马酸丙酚替诺福韦片治疗CHB患者,可有效减轻机体氧化应激,调节CD4⁺CD25⁺调节性T细胞和血清MMP-1、MMP-2、TIMP-1水平。     

Resistance to respiratory syncytial virus (RSV) challenge induced by infection with a vaccinia virus recombinant expressing the RSV M2 protein (Vac-M2) is mediated by CD8+ T cells, while that induced by Vac-F or Vac-G recombinants is mediated by antibodies.

It was previously demonstrated that the vaccinia virus recombinants expressing the respiratory syncytial virus (RSV) F, G, or M2 (also designated as 22K) protein (Vac-F, Vac-G, or Vac-M2, respectively) induced almost complete resistance to RSV challenge in BALB/c mice. In the present study, we sought to identify the humoral and/or cellular mediators of this resistance. Mice were immunized by infection with a single recombinant vaccinia virus and were subsequently given a monoclonal antibody directed against CD4+ or CD8+ T cells or gamma interferon (IFN-gamma) to cause depletion of effector T cells or IFN-gamma, respectively, at the time of RSV challenge (10 days after immunization). Mice immunized with Vac-F or Vac-G were completely or almost completely resistant to RSV challenge after depletion of both CD4+ and CD8+ T cells prior to challenge, indicating that these cells were not required at the time of virus challenge for expression of resistance to RSV infection induced by the recombinants. In contrast, the high level of protection of mice immunized with Vac-M2 was completely abrogated by depletion of CD8+ T cells, whereas depletion of CD4+ T cells or IFN-gamma resulted in intermediate levels of resistance. These results demonstrate that antibodies are sufficient to mediate the resistance to RSV induced by the F and G proteins, whereas the resistance induced by the M2 protein is mediated primarily by CD8+ T cells, with CD4+ T cells and IFN-gamma also contributing to resistance.