Newcastle disease virus-based MERS-CoV candidate vaccine elicits high-level and lasting neutralizing antibodies in Bactrian camels

Middle East respiratory syndrome coronavirus （MERS-CoV）, a member of the Coronavifidae family, is the causative pathogen for MERS that is characterized by high fever, pneumonia, acute respiratory distress syndrome （ARDS）, as well as extrapul- monary manifestations. Currently, there are no approved treatment regimens or vaccines for MERS. Here~ we generated recombinant nonvirulent Newcastle disease virus （NDV） LaSota strain expressing MERS-CoV S protein （designated as rLa- MERS-S）, and evaluated its immunogenicity in mice and Bactrian camels. The results revealed that rLa-MERS-S showed similar growth properties to those of LaSota in embryonated chicken eggs, while animal immunization studies showed that rLa-MERS-S induced MERS-CoV neutralizing antibodies in mice and camels. Our findings suggest that recombinant rLa- MERS-S may be a potential MERS-CoV veterinary vaccine candidate for camels and other animals affected by MERS.

Transforming growth factor -β neutralizing antibodies inhibit subretinal fibrosis in a mouse model

AIM:To determine the involvement of the transforming growth factor(TGF)-β with the development of experimental subretinal fibrosis in a mouse model.· METHODS:Subretinal fibrosis was induced by subretinal injection of macrophage-rich peritoneal exudate cells(PECs) and the local expression of TGF-β isoforms was assessed by quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR) and enzyme-linked immunosorbent assay(ELISA) at various time points.In addition,we investigated the effect of TFG-β-neutralizing antibodies(TGF-β NAb) on subretinal fibrosis development.· RESULTS:TGF-β1 and TGF-β2 mRNA level was significantly elevated at day 2 after subretinal fibrosis induction and increased further to 5 and 6.5-fold respectively at day 5,reaching the peak.TGF-β3 mRNA was not detected in the present study.The result of ELSIA showed that active TGF-β1 and TGF-β2 levels were upregulated to 10-fold approximately,while total TGF-β1 and TGF-β2 levels were even upregulated more than 10-fold and more than 20-fold respectively in subretinal fibrosis mice in comparison with na?觙ve mice at day 5.TGF-β NAb resulted in a reduced subretinal fibrosis areas by 65% compared to animals from control group at day 7.· CONCLUSION:Our results indicate that TGF-β signaling may contribute to the pathogenesis of subretinal fibrogenesis and TGF-β inhibition may provide an effective,novel treatment of advanced and late-stage neovascular age-related macular degeneration.·

Epitope Analysis of Anti-SARS-CoV-2 Neutralizing Antibodies

Coronavirus disease 2019 is threatening thousands of millions of people around the world.In the absence of specific and highly effective medicines,the treatment of infected persons is still very challenging.As therapeutics,neutralizing antibodies(NAbs)have great potential.Many NAbs have been reported,and most target various regions on the receptor-binding domain of the spike(S)protein,or the N-terminal domain.Several NAbs and NAb cocktails have been authorized for emergency use,and more arc in clinical trials or are under development.In this review,considering the angle of binding epitopes on the S protein,we summarize the functions and the underlying mechanisms of a set of well-recognized NAbs and provide guidance for vaccine design and the combinatorial use of these antibodies.In addition,we review the NAbs and NAb cocktails that have been approved for emergency use and discuss the effectiveness of these NAbs for combating severe acute respiratory syndrome coronavirus 2 mutants.

Safe and Objective Assay of Enterovirus 71 Neutralizing Antibodies via Pseudovirus

Current serum neutralization assays based on the inhibition of the cytopathic effect（Nt-CPE） need to ma nipulate live viruses, which are time-consuming, labor-intensive, and have the potential exposure to infectious agents, so a safe and objective assay via pseudovirus for the fast and efficient detection of enterovirus 71（EV71） neutralizing antibodies was developed. First, we generated EV71 pseudovirus containing firefly luciferase gene in place of the capsid gene P1 in EV71 genome. Vero cells infected with 200 CCID50（50% cell culture infective dose） of EV71 pseudovirus for 24 h were found to have the best performance. Seval sera were measured by EV71 pseudoparticle neutralization assay（Nt-PPN） and the conventional serological method Nt-CPE. Neutralizing antibody titers measured by Nt-PPN and those obtained by Nt-CPE demonstrate a high correlation between the two methods. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of EV71 neutralizing anti bodies. This method can be used for detecting neutralizing antibodies of other picornaviruses, such as hepatitis A vi rus（HAV） and coxsackievirus 16（CVA16）, and make it possible to determine whether there is cross-reactivity be tween EV71 and CVA16.

Neutralizing Antibody Responses against Five SARS-CoV-2 Variants and T Lymphocyte Change after Vaccine Breakthrough Infections from the SARS-CoV-2 Omicron BA.1 Variant in Tianjin, China: A Prospective Study

Objective To investigate whether Omicron BA.1 breakthrough infection after receiving the SARS-CoV-2 vaccine could create a strong immunity barrier.Methods Blood samples were collected at two different time points from 124 Omicron BA.1 breakthrough infected patients and 124 controls matched for age,gender,and vaccination profile.Live virus-neutralizing antibodies against five SARS-CoV-2 variants,including WT,Gamma,Beta,Delta,and Omicron BA.1,and T-lymphocyte lymphocyte counts in both groups were measured and statistically analyzed.Results The neutralizing antibody titers against five different variants of SARS-CoV-2 were significantly increased in the vaccinated population infected with the Omicron BA.1 variant at 3 months after infection,but mainly increased the antibody level against the WT strain,and the antibody against the Omicron strain was the lowest.The neutralizing antibody level decreased rapidly 6 months after infection.The T-lymphocyte cell counts of patients with mild and moderate disease recovered at 3 months and completely returned to the normal state at 6 months.Conclusion Omicron BA.1 breakthrough infection mainly evoked humoral immune memory in the original strain after vaccination and hardly produced neutralizing antibodies specific to Omicron BA.1.Neutralizing antibodies against the different strains declined rapidly and showed features similar to those of influenza.Thus,T-lymphocytes may play an important role in recovery.

A SARS-CoV-2 neutralizing antibody discovery by single cell sequencing and molecular modeling

Although vaccines have been developed,mutations of SARS-CoV-2,especially the dominant B.1.617.2(delta)and B.1.529(omicron)strains with more than 30 mutations on their spike protein,have caused a significant decline in prophylaxis,calling for the need for drug improvement.Antibodies are drugs preferentially used in infectious diseases and are easy to get from immunized organisms.The current study combined molecular modeling and single memory B cell sequencing to assess candidate sequences before experiments,providing a strategy for the fabrication of SARS-CoV-2 neutralizing antibodies.A total of 128 sequences were obtained after sequencing 196 memory B cells,and 42 sequences were left after merging extremely similar ones and discarding incomplete ones,followed by homology modeling of the antibody variable region.Thirteen candidate sequences were expressed,of which three were tested positive for receptor binding domain recognition but only one was confirmed as having broad neutralization against several SARS-CoV-2 variants.The current study successfully obtained a SARS-CoV-2 antibody with broad neutralizing abilities and provided a strategy for antibody development in emerging infectious diseases using single memory B cell BCR sequencing and computer assistance in antibody fabrication.

Two antibodies show broad,synergistic neutralization against SARS-CoV-2 variants by inducing conformational change within the RBD

Continual evolution of the severe acute respiratory syndrome coronavirus(SARS-CoV-2)virus has allowed for its gradual evasion of neutralizing antibodies(nAbs)produced in response to natural infection or vaccination.The rapid nature of these changes has incited a need for the development of superior broad nAbs(bnAbs)and/or the rational design of an antibody cocktail that can protect against the mutated virus strain.Here,we report two angiotensin-converting enzyme 2 competing nAbs—8H12 and 3E2—with synergistic neutralization but evaded by some Omicron subvariants.Cryo-electron microscopy reveals the two nAbs synergistic neutralizing virus through a rigorous pairing permitted by rearrangement of the 472-489 loop in the receptor-binding domain to avoid steric clashing.Bispecific antibodies based on these two nAbs tremendously extend the neutralizing breadth and restore neutralization against recent variants including currently dominant XBB.1.5.Together,these findings expand our understanding of the potential strategies for the neutralization of SARS-CoV-2 variants toward the design of broad-acting antibody therapeutics and vaccines.

Recent Developments in SARS-CoV-2 Neutralizing Antibody Detection Methods

The ongoing Coronavirus disease 19 pandemic has likely changed the world in ways not seen in the past.Neutralizing antibody(NAb)assays play an important role in the management of the severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)outbreak.Using these tools,we can assess the presence and duration of antibody-mediated protection in naturally infected individuals,screen convalescent plasma preparations for donation,test the efficacy of immunotherapy,and analyze NAb titers and persistence after vaccination to predict vaccine-induced protective effects.This review briefly summarizes the various methods used for the detection of SARS-CoV-2 NAbs and compares their advantages and disadvantages to facilitate their development and clinical application.

Neurotoxic role of interleukin-17 in neural stem cell differentiation after intracerebral hemorrhage

Interleukin 17(IL-17)and its main producer,T cell receptorγδcells,have neurotoxic effects in the pathogenesis of intracerebral hemorrhage(ICH),aggravating brain injuries.To investigate the correlation between IL-17 and ICH,we dynamically screened serum IL-17 concentrations using enzyme-linked immunosorbent assay and explored the clinical values of IL-17 in ICH patients.There was a significant negative correlation between serum IL-17 level and neurological recovery status in ICH patients(r=–0.498,P<0.01).To study the neurotoxic role of IL-17,C57 BL/6 mice were used to establish an ICH model by injecting autologous blood into the caudate nucleus.Subsequently,the mice were treated with mouse neural stem cells(NSCs)and/or IL-17 neutralizing antibody for 72 hours.Flow cytometry,brain water content detection,Nissl staining,and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling results indicated that NSC transplantation significantly reduced IL-17 expression in peri-hematoma tissue,but there was no difference in T cell receptorγδcells.Compared with the ICH group,there were fewer apoptotic bodies and more Nissl bodies in the ICH+NSC group and the ICH+NSC+IL-17 group.To investigate the potential effect of IL-17 on directional differentiation of NSCs,we cultured mouse NSCs(NE-4 C)alone or co-cultured them with T cell receptorγδcells,which were isolated from mouse peripheral blood mononuclear cells,for 7 days.The results of western blot assays revealed that IL-17 secreted by T cell receptorγδcells reduced the differentiation of NSCs into astrocytes and neurons,while IL-17 neutralization relieved the inhibition of directional differentiation into astrocytes rather than neurons.In conclusion,serum IL-17 levels were elevated in the early stage of ICH and were negatively correlated with outcome in ICH patients.Animal experiments and cytological investigations therefore demonstrated that IL-17 probably has neurotoxic roles in ICH because of its inhibitory effects on the directional differentiation of NSCs.The application of IL-17 neutralizing antibody may promote the directional differentiation of NSCs into astrocytes.This study was approved by the Clinical Research Ethics Committee of Anhui Medical University of China(For human study:Approval No.20170135)in December 2016.All animal handling and experimentation were reviewed and approved by the Institutional Animal Care and Use Committee of Anhui Medical University(approval No.20180248)in December 2017.

Convalescent plasma: A potential therapeutic option for COVID-19 patients

The new coronavirus disease(COVID-19)outbreak has challenged us to take unprecedented steps to bring this pandemic under control.In view of the urgency of this situation,convalescent plasma which was used in previous coronavirus outbreaks has emerged as one of the treatment options in this current pandemic.This is mainly due to the fact that convalescent plasma has been studied in a few case series with promising outcomes.In addition,on-going large clinical trials aimed to further evaluate the effectiveness,safety,and optimal dosage,duration and timing of administration of convalescent plasma are indeed revealing a certain level of promising results.Therefore,this article aims to provide an overview of possible mechanisms of actions of convalescent plasma,its benefits and its level of usage safeness by summarizing the existing evidence on the use of convalescent plasma in COVID-19 patients.

Immunization Schedule of Liposomal Rabies Vaccine in Animals

The standard rabies vaccines recommended by WHO include Essen regimen, the Thai Red Cross two-site ID regimen and the eight-site ID regimen, and so on. The present schedules of rabies vaccine are all laborious and time consuming. We developed a new rabies vaccine with liposome as adjuvant（LipoRabV） and found that liposome could facilitate the inactivated rabies vaccine（RabV） to induce the more vigorous production of rabies virus neutralizing antibody（RVNA） in BALB/c mice and beagles. We established preliminary pre- and post-exposure prophylaxis schedules for LipoRabV. LipoRabV（0/14） could elicit similar RVNA level as RabV（0/7/28） by pre-exposure prophylaxis schedules in mice and beagles. LipoRabV（0/3/14） could elicit higher RVNA level vs. RabV（0/3/7/14/28） in BALB/c. The data indicate that the three-shot liposome-enhanced rabies vaccine could achieve a higher protection rate（survival rate 56.2%） by post-exposure prophylaxis compared with that of the RabV group（survival rate 40.6%） in mice. The data also indicate that the three-inoculation liposome-enhanced rabies vaccine could achieve a survival rate of 80.0% vs. RabV（70.0%） by post-exposure prophylaxis in beagles. The results show that the immunization schedule for LipoRabV could be preliminarily determined at 0 and 14 d for pre-exposure prophylaxis and at 0, 3 and 14 d for post-exposure prophylaxis.

Kinetic Characteristics of Neutralizing Antibody Responses Vary among Patients with COVID-19

Objective The coronavirus disease 2019(COVID-19) pandemic continues to present a major challenge to public health. Vaccine development requires an understanding of the kinetics of neutralizing antibody(NAb) responses to severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Methods In total, 605 serum samples from 125 COVID-19 patients(from January 1 to March 14, 2020)varying in age, sex, severity of symptoms, and presence of underlying diseases were collected, and antibody titers were measured using a micro-neutralization assay with wild-type SARS-CoV-2.Results NAbs were detectable approximately 10 days post-onset(dpo) of symptoms and peaked at approximately 20 dpo. The NAb levels were slightly higher in young males and severe cases, while no significant difference was observed for the other classifications. In follow-up cases, the NAb titer had increased or stabilized in 18 cases, whereas it had decreased in 26 cases, and in one case NAbs were undetectable at the end of our observation. Although a decreasing trend in NAb titer was observed in many cases, the NAb level was generally still protective.Conclusion We demonstrated that NAb levels vary among all categories of COVID-19 patients. Longterm studies are needed to determine the longevity and protective efficiency of NAbs induced by SARS-CoV-2.

Efficient Humoral and Cellular Immune Responses Induced by a Chimeric Virus-like Particle Displaying the Epitope of EV71 without Adjuvant

Objective To eliminate the side effects of aluminum adjuvant and His-tag,we constructed chimeric VLPs displaying the epitope of EV71（SP70） without His-tagged.Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.Methods The fusion protein was constructed by inserting SP70 into the MIR of truncated HBc Ag sequence,expressed in E.Coli,and purified through ion exchange chromatography and density gradient centrifugation.Mice were immunized with the VLPs and sera were collected afterwards.The specific antibody titers,Ig G subtypes and neutralizing efficacy were detected by ELISA,neutralization assay,and EV71 lethal challenge.IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.Results HBc-SP70 proteins can self-assemble into empty VLPs.After immunization with HBc-SP70 VLPs,the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge.There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not.The specific Ig G subtypes were mainly IgG1 and IgG2 b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.Conclusion The fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation.In the absence of adjuvant,they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant.Furthermore,the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.

Neutralizing Antibody Titer Test of Ebola Recombinant Protein Vaccine and Gene Vector Vaccine pVR-GP-FC

Objective In previous studies, we immunized mice with Ebola recombinant protein vaccine and gene vector vaccine. Both stimulated high levels of humoral immunity. In this work, we constructed a pseudovirus containing Ebola membrane proteins to verify whether the two immunization strategies can induce neutralizing antibodies in mice. Methods A pseudovirus containing an Ebola virus membrane protein based on the HIV-1 viral gene sequence was constructed and evaluated using a known neutralizing antibody. The titer of the neutralizing antibody in the sera of mice immunized with the recombinant protein and the gene vector vaccine was examined using a neutralization test. Results Ebola pseudovirus was successfully prepared and applied for neutralizing antibody detection. Immunological experiments showed that recombinant protein GP-Fc and gene vaccine pVR-modGP-Fc had good immunogenicity. The titer of the bound antibody in the serum after 8 weeks of immunization in mice was more than 1：105, and the recombinant protein induced greater humoral immunity. The results of the neutralization test based on the Ebola pseudovirus system demonstrated that both vaccines induced production of protective antibodies, while the gene vaccine induced a higher titer of neutralizing antibodies. Conclusion An Ebola pseudovirus detection system was successfully established and used to evaluate two Ebola vaccines. Both produced good immunogenicity. The findings lay the foundation for the development of new Ebola vaccines and screening for neutralizing monoclonal antibodies.

Polyfunctional T Cell and Neutralizing Antibody Responses to ACAM2000TM Smallpox Vaccine Immunization in Primary-Vaccinated Individuals

Smallpox eradication was successful via prophylactic administration of live attenuated vaccinia virus. As a result of the discontinuation of the smallpox immunization program, many individuals are now susceptible to smallpox virus infection should it be used as a biological weapon. Presently, only individuals at high risk for exposure are required to receive smallpox vaccine, such as laboratory personnel that handle variola/vaccinia virus. This study endeavored to investigate a one-year period of vaccinia virus-specific T cell responses using polychromatic flow cytometry and neutralizing (Nt) antibody responses using plaque reduction neutralization test (PRNT) in individuals receiving primary immunization (n = 5) with ACAM2000TM smallpox vaccine. Functional and phenotypic profiles of vaccinia virus-specific T cell responses were characterized. Each single functional measurement {CD107a/b expression, production of interferon g (IFN-g), macrophage inflammatory protein 1b (MIP-1b), interleukin 2 (IL-2), and tumor necrosis factor a (TNF-a)} demonstrated that vaccinia virus-specific CD8+ T cells were functional at least one time point after vaccination (p ≤ 0.05). However, vaccinia virus-specific CD4+ T cells were functional only for MIP-1b production (p ≤ 0.05). Vaccinia virus-specific CD8+ T cells induced in these individuals showed increased polyfunctionality in at least 2 phenotypes relative to pre-vaccination (p ≤ 0.05). Although only three of five individuals (60%) showed positive Nt antibody (titer ≥ 20) at first month after vaccination, all five individuals (100%) demonstrated Nt antibody at 2 months, post-immunization. Interestingly, all vaccinees could retain the Nt antibody for 6 months after primary vaccination. In conclusion, ACAM2000TM smallpox vaccine induced both polyfunctional T cell-and Nt antibody-responses in primary immunized individuals.

Broad strategies for neutralizing SARS-CoV-2 and other human coronaviruses with monoclonal antibodies

Antibody therapeutics and vaccines for coronavirus disease 2019(COVID-19)have been approved in many countries,with most being developed based on the original strain of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).SARS-CoV-2 has an exceptional ability to mutate under the pressure of host immunity,especially the immune-dominant spike protein of the virus,which is the target of both antibody drugs and vaccines.Given the continuous evolution of the virus and the identification of critical mutation sites,the World Health Organization(WHO)has named 5 variants of concern(VOCs):4 are previously circulating VOCs,and 1 is currently circulating(Omicron).Due to multiple mutations in the spike protein,the recently emerged Omicron and descendent lineages have been shown to have the strongest ability to evade the neutralizing antibody(NAb)effects of current antibody drugs and vaccines.The development and characterization of broadly neutralizing antibodies(bNAbs)will provide broad strategies for the control of the sophisticated virus SARS-CoV-2.In this review,we describe how the virus evolves to escape NAbs and the potential neutralization mechanisms that associated with bNAbs.We also summarize progress in the development of bNAbs against SARS-CoV-2,human coronaviruses(CoVs)and other emerging pathogens and highlight their scientific and clinical significance.

Inactivated Pseudomonas PE(△Ⅲ)exotoxin fused to neutralizing epitopes of PEDV S proteins produces a specific immune response in mice

Porcine epidemic diarrhea(PED)caused by the porcine epidemic diarrhea virus(PEDV),is a severe infectious and devastating swine disease that leads to serious economic losses in the swine industry worldwide.An increased number of PED cases caused by variant PEDV have been reported in many countries since 2010.S protein is the main immunogenic protein containing some B-cell epitopes that can induce neutralizing antibodies of PEDV.In this study,the construction,expression and purification of Pseudomonas aeruginosa exotoxin A(PE)without domain Ⅲ(PE△Ⅲ)as a vector was performed for the delivery of PEDV S-A or S-B.PE(△Ⅲ)PEDV S-A and PE(△Ⅲ)PEDV S-B recombinant proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis.The immunogenicity of PEDV S-A and PEDV S-B subunit vaccines were evaluated in mice.The results showed that PEDV-S-B vaccine could not only induce specific humoral and Th1 type-dominant cellular immune responses,but also stimulate PEDV-specific mucosal immune responses in mice.PEDV-S-B subunit vaccine is a novel candidate mucosal vaccine against PEDV infection.

Symptomatic and Asymptomatic SARS-CoV-2 Infection and Follow-up of Neutralizing Antibody Levels

Objective To investigate neutralizing antibody levels in symptomatic and asymptomatic patients with coronavirus disease 2019(COVID-19)at 6 and 10 months after disease onset.Methods Blood samples were collected at three different time points from 27 asymptomatic individuals and 69 symptomatic patients infected with severe acute respiratory syndrome coronavirus 2(SARS-Co V-2).Virus-neutralizing antibody titers against SARS-CoV-2 in both groups were measured and statistically analyzed.Results The symptomatic and asymptomatic groups had higher neutralizing antibodies at 3 months and 1–2 months post polymerase chain reaction confirmation,respectively.However,neutralizing antibodies in both groups dropped significantly to lower levels at 6 months post-PCR confirmation.Conclusion Continued monitoring of symptomatic and asymptomatic individuals with COVID-19 is key to controlling the infection.

Insights into the virologic and immunologic features of SARS-COV-2

The host immunity is crucial in determining the clinical course and prognosis of coronavirus disease 2019,where some systemic and severe manifestations are associated with excessive or suboptimal responses.Several antigenic epitopes in spike,nucleocapsid and membrane proteins of severe acute respiratory syndrome coronavirus 2 are targeted by the immune system,and a robust response with innate and adaptive components develops in infected individuals.High titer neutralizing antibodies and a balanced T cell response appears to constitute the optimal immune response to severe acute respiratory syndrome coronavirus 2,where innate and mucosal defenses also contribute significantly.Following exposure,immunological memory seems to develop and be maintained for substantial periods.Here,we provide an overview of the main aspects in antiviral immunity involving innate and adaptive responses with insights into virus structure,individual variations pertaining to disease severity as well as long-term protective immunity expected to be attained by vaccination.

Anemia in ESKD Patient (End-Stage Kidney Disease): The Rare Cause Is in the Forefront

We reported a case of severe anemia in a patient with end-stage kidney disease (ESKD) on dialysis. The anemia developed when the patient is switched from hemodialysis (HD) to peritoneal dialysis (PD) when the intra-venous erythropoietin stimulating agent (ESA, Epogen) was changed into subcutaneous injection of darbepoetin. The patient’s hemoglobin dropped 2 grams in about two months during this period. Extensive work-up including, bleeding disorders, hemolysis, iron deficiency, infections including CMV, Epstein-Bar virus, parvo-19 virus infection were unrevealing The anti-Epogen neutralizing antibodies were not measured due to unavailability. Bone marrow biopsy and aspirate were negative for infiltrations or myelodysplastic syndrome (MDS). The leukocyte and platelet counts were normal. Even though anti-ESA antibodies were not measured in this case, all tentative causes of his anemia were excluded by laboratory investigations. The patient’s anemia was treated symptomatically with blood transfusion and discontinuation of the ESA treatment. He made a remarkable recovery.