The relationship of microvascular inflammation with antibody-mediated rejection in kidney transplantation.

Microvascular inflammation (MVI) is a key diagnostic feature of antibody-mediated rejection (AMR); however, recipients without donor-specific antibodies (DSA) defy etiologic classification using C4d staining of peritubular capillaries (C4dptc) and conventional DSA assignment. We evaluated MVI ≥ 2 (Banff g + ptc ≥ 2) using Banff 2019 AMR (independent of MVI ≥ 2 but including C4dptc) with unconventional endothelial C4d staining of glomerular capillaries (C4dglom) and - arterial endothelium and/or intima (C4dart) using tissue immunoperoxidase, shared-eplet and subthreshold DSA (median fluorescence intensity, [MFI] 100-499), and capillary ultrastructure from 3398 kidney transplant samples for evidence of AMR. MVI ≥ 2 (n = 202 biopsies) from 149 kidneys (12.4% prevalence) correlated with DSA+, C4dptc+, C4dglom+, Banff cg, i, t, ti scores, serum creatinine, proteinuria, and graft failure compared with 202 propensity score matched normal controls. The laboratory reported DSA- MVI ≥ 2 (MFI ≥500) occurred in 34.7%; however, subthreshold (28.6%), eplet-directed (51.4%), and/or misclassified anti-Human leukocyte antigen (HLA) DSA (12.9%) were identified in 67.1% by forensic reanalysis, with vascular C4d+ staining in 67.1%, and endothelial abnormalities in 57.1%, totaling 87.1%. Etiologic analysis attributed 62.9% to AMR (77.8% for MVI with negative reported DSA [DSA- MVI ≥2] with glomerulitis) and pure T cellular rejection in 37.1%. C4dptc-DSA- MVI ≥ 2 was unrecognized AMR in 48.0%. Functional outcomes and graft survival were comparable to normal controls. We concluded that DSA- MVI ≥ 2 frequently signified a mild "borderline" phenotype of AMR which was recognizable using novel serologic and pathological techniques.

Escalated complement activation during hospitalization is associated with higher risk of 60-day mortality in SARS-CoV-2-infected patients.

BACKGROUND: The complement system, an upstream recognition system of innate immunity, is activated upon SARS-CoV-2 infection. To gain a deeper understanding of the extent and duration of this activation, we investigated complement activation profiles during the acute phase of COVID-19, its persistence post-recovery and dynamic changes in relation to disease severity. METHODS: Serial blood samples were obtained from two cohorts of hospitalized COVID-19 patients (n = 457). Systemic complement activation products reflecting classical/lectin (C4d), alternative (C3bBbP), common (C3bc) and terminal pathway (TCC and C5a) were measured during hospitalization (admission, days 3-5 and days 7-10), at 3 months and after 1 year. Levels of activation and temporal profiles during hospitalization were related to disease severity defined as respiratory failure (PO2/FiO2 ratio <26.6 kPa) and/or admission to intensive care unit, 60-day total mortality and pulmonary pathology after 3 months. FINDINGS: During hospitalization, TCC, C4d, C3bc, C3bBbP and C5a were significantly elevated compared to healthy controls. Severely ill patients had significantly higher levels of TCC and C4d (p < 0.001), compared to patients with moderate COVID-19. Escalated levels of TCC and C4d during hospitalization were associated with a higher risk of 60-day mortality (p < 0.001), and C4d levels were additionally associated with chest CT changes at 3 months (p < 0.001). At 3 months and 1 year, we observed consistently elevated levels of most complement activation products compared to controls. CONCLUSION: Hospitalized COVID-19 patients display prominent and long-lasting systemic complement activation. Optimal targeting of the system may be achieved through enhanced risk stratification and closer monitoring of in-hospital changes of complement activation products.

Association between urinary C4d levels and disease progression in IgA nephropathy.

BACKGROUND: C4d mesangial deposition, a hallmark of lectin pathway activation in immunoglobulin A nephropathy (IgAN), has been shown to be associated with risk of kidney failure. To date, the relationship between urinary C4d and renal outcome remain unelucidated. METHODS: A total of 508 patients with biopsy-proven IgAN were enrolled in this study, whose baseline urine samples at the time of biopsy were collected and the levels of urinary C4d were quantified by enzyme-linked immunosorbent assay. The time-averaged C4d (TA-C4d) and the change in proteinuria were measured in sequential urine samples obtained from IgAN patients. The kidney progression event was defined as a 50% estimated glomerular filtration rate (eGFR) decline or end-stage kidney disease or death. RESULTS: After a median follow-up of 36 months, 70 (13.8%) of the participants reached the kidney progression event. Higher levels of urinary C4d/Ucr were found to be associated with decreased eGFR, massive proteinuria, lower serum albumin levels, hypertension, and severe Oxford E and T scores. Upon adjusting for traditional risk factors (including demographics, eGFR, proteinuria, hypertension, Oxford pathologic score and immunosuppressive therapy), elevated levels of urinary C4d/Ucr were independently associated with an increased risk of chronic kidney disease progression [adjusted hazard ratio (HR) per standard deviation increment of log-transformed C4d/Ucr: 1.46; 95% CI 1.04-2.06; P = .030]. In reference to the low C4d group, the risk of poor renal outcome increased for the high C4d group (adjusted HR 1.93; 95% CI 1.05-3.54; P = .033). Additionally, a low baseline C4d level was independently associated with a favorable proteinuria response to immunosuppressive therapy at 3 months (adjusted relative risk 2.20; 95% CI 1.04-4.63, P = .038). CONCLUSION: The urinary C4d, serving as a non-invasive biomarker, is associated with the progression of IgAN and holds the potential to predict proteinuria response in this disease.

Urinary C4d and progression of kidney disease in IgA vasculitis.

BACKGROUND: Immunoglobulin A (IgA) vasculitis nephritis (IgAVN) is the most common secondary IgA nephropathy (IgAN). Urinary C4d have been identified associated with the development and progression in primary IgAN; however, its role in kidney disease progression of IgAVN is still unclear. METHODS: This study enrolled 139 patients with IgAVN, 18 healthy subjects, 23 focal segmental glomerulosclerosis patients and 38 IgAN patients. Urinary C4d levels at kidney biopsy were measured using enzyme-linked immunosorbent assay. The association between urinary C4d/creatinine and kidney disease progression event, defined as 40% estimated glomerular filtration rate decline or end-stage kidney disease, was assessed using Cox proportional hazards models and restricted cubic splines. RESULTS: The levels of urinary C4d/creatinine (Cr) in IgAVN and IgAN patients were higher than in healthy controls. Higher levels of urinary C4d/Cr were associated with higher proteinuria and severe Oxford C lesions, and glomerular C4d deposition. After a median follow-up of 52.79 months, 18 (12.95%) participants reached composite kidney disease progression event. The risk of kidney disease progression event was higher with higher levels of Ln(urinary C4d/Cr). After adjustment for clinical data, higher levels of urinary C4d/Cr were associated with kidney disease progression in IgAVN [per Ln-transformed urinary C4d/Cr, hazard ratio 1.573, 95% confidence interval (CI) 1.101-2.245; P = .013]. Compared with the lower C4d/Cr group, the hazard ratio was 5.539 (95% CI 1.135-27.035; P = .034) for the higher levels group. CONCLUSIONS: Higher levels of urinary C4d/Cr were associated with kidney disease progression event in patients with IgAVN.

Complement C4d as a biomarker for systemic lupus erythematosus and lupus nephritis.

Background: Increasing studies in the last decade have led to the widespread understanding that C4d, a split product of complement component 4 (C4), is a potential biomarker for systemic lupus erythematosus (SLE) and lupus nephritis (LN).Purpose: The aim of this review is to summarize the highlights of studies investigating the use of C4d as a biomarker for diagnosing and monitoring SLE and LN patients.Data collection: we searched PubMed/Medline and Wanfang databases using the terms "C4d and systemic lupus erythematosus", "C4d and lupus nephritis", and "Complement C4d".Results: The deposition of C4d on circulating blood cells has been shown in several clinical studies to be a potential diagnostic marker that can be used to monitor patients with SLE. In addition, C4d deposits on circulating blood cells may be a helpful diagnostic marker for LN, one of the most severe complications of SLE. Meanwhile, studies utilizing renal biopsy specimens have indicated that C4d deposition in the renal peritubular capillaries of LN patients may predict more severe LN or a worse patient prognosis. Generally, a high plasma C4d level and a high plasma C4d/C4 ratio may also be promising indicators that can be used to monitor patients with SLE and LN.Conclusions: C4d detection may be a novel strategy for further clinical prediction and therapy.

Antibody-Mediated Rejection in Liver Transplantation: Immuno-Pathological Characteristics and Long-Term Follow-Up.

The diagnosis of liver antibody-mediated rejection (AMR) is challenging and likely under-recognized. The association of AMR with donor-specific antibodies (DSA), and its clinical course in relation to pathologic findings and treatment are ill defined. We identified cases of liver AMR by following the criteria outlined by the 2016 Banff Working Group. Patient demographics, native liver disease, histopathologic findings, treatment type, clinical outcome, and transaminase levels during AMR diagnosis, treatment, and resolution were determined. Patients (n = 8) with AMR average age was 55.2 years (range: 19-68). Seven of eight cases met the Banff criteria for AMR. Personalized treatment regimens consisted of optimization of immunosuppression, intravenous pulse steroids, plasmapheresis, IVIG, rituximab, and bortezomib. Five patients experienced complete resolution of AMR, return of transaminases to baseline, and decreased DSA at long-term follow-up. One patient developed chronic AMR and two patients required re-transplantation. Follow-up after AMR diagnosis ranged from one to 11 years. Because AMR can present at any time, crossmatch, early biopsy, and routine monitoring of DSA levels should be implemented following transaminase elevation to recognize AMR. Furthermore, treatment should be immediately implemented to reverse AMR and prevent graft failure, chronic damage, re-transplantation, and possibly mortality.

Diagnosis of autoimmune bullous dermatoses: Comparative analysis of immunohistochemical staining using C4d, C3d, IgG, and IgG4 in lesional tissues and perilesional frozen skin samples.

Immunohistochemical staining with immunoglobulins and complements may aid the diagnosis of patients whose clinical and histological findings are consistent with autoimmune bullous dermatoses (AIBD). We aimed to investigate the diagnostic value of immunohistochemical markers in lesional biopsy and perilesional frozen samples in AIBD. We included 136 cases from whom lesional biopsies and perilesional samples for direct immunofluorescence (DIF) examination were collected with a preliminary diagnosis of AIBD between January 2019 and January 2023. All diagnoses were reconfirmed by evaluating the clinical, histopathological, and serological findings and DIF results (C3, IgG, IgA, or IgM positivity compatible with the clinical diagnosis) altogether, although DIF results were considered a priority. After confirming the diagnoses, the samples were categorized as AIBD or the others. The perilesional tissues obtained for DIF simultaneously with skin biopsy and stored at -80 °C were thawed, and FFPE tissues were prepared. We performed immunohistochemical staining (C4d, C3d, IgG, and IgG4) on FFPE tissues of both lesional and perilesional samples. Strong, linear, or granular staining patterns at the dermoepidermal junction or the intraepidermal blistering space were considered positive in line with the diagnosis of the case. Cases other than AIBD were used as negative control tissues to assess the specificity of immunohistochemical markers. Of the 136 cases, 52 were diagnosed with AIBD. In lesional samples, the sensitivity of C4d, C3d, IgG, and IgG4 was 80.6 %, 69.4 %, 75 %, and 5.7 % with corresponding specificity of 100 %, 98.7 %, 89.6 %, and 97.4 %, respectively in pemphigoid diseases compared to a sensitivity of 18.2 %, 9.1 %, 70 %, and 9.1 % and specificity of 98.7 %, 100 %, 89.6 %, and 97.4 %, respectively in pemphigus diseases. In frozen samples, we detected expression in a limited number of cases. The sensitivity of C4d, C3d, IgG, and IgG4 was 8.7 %, 2.2 %, 19.4 %, and 2.2 %, with corresponding specificity of 100 %, 100 %, 98.5 %, and 98.6, respectively. There was a none to slight concordance rate between the IHC results of lesional tissues and perilesional frozen samples. Kappa coefficients for C4d, C3d, IgG, and IgG4 were 0.120 (P = 0.029), 0.111 (P = 0.050), 0.203 (P = 0.003), and - 0.15 (P = 0.846), respectively. Immunohistochemical staining with C4d, C3d, IgG, and IgG4 on biopsy samples collected from lesions may guide the diagnosis of AIBD, thereby eliminating the need for an additional biopsy and accelerating the diagnostic process.

Evaluation of C4d expression and staining patterns by immunohistochemistry in renal biopsy samples with focal segmental glomerulosclerosis and minimal change disease.

INTRODUCTION: C4d is an activation product of lectin pathway of complement. Glomerular deposition of C4d is associated with poor prognosis in different types of immune-related glomerulonephritis. The present study was conducted to investigate expression level of C4d and its staining pattern in renal biopsy of patients with focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) by immunohistochemistry method. MATERIALS AND METHODS: In this retrospective cross-sectional study, renal biopsy specimens from 46 samples of MCD, 47 samples of FSGS, and 15 samples without glomerular disease as the controls, were subjected to immunohistochemistry staining with C4d. Demographic characteristics and information obtained from light and electron microscopy (EM) of patients were also extracted from their files. RESULTS: C4d positive staining was observed in 97.9 % of FSGS and 43.5 % of MCD samples, which showed a statistically significant difference (P < 0.001). The sensitivity and specificity of C4d expression for diagnosing FSGS were 97.9 % and 56.5 %, respectively. There was no significant correlation between C4d expression and any of the light and electron microscopy findings, including presence of foam cells, mesangial matrix expansion, interstitial fibrosis and tubular atrophy, and basement membrane changes in MCD patients. Also, no significant correlation was observed between C4d expression and clinical symptoms of proteinuria or prolonged high level of creatinine in patients with MCD. DISCUSSION AND CONCLUSION: The expression of C4d marker had a good sensitivity and negative predictive value in the diagnosis of FSGS.

Accurate Visualization of C4d Complement Fragment in Immunohistochemistry by C-Terminal Linear Neoepitope-Specific Antibodies.

C4d is the end degradation product of activated complement component C4b that appears during the early steps of the classical and lectin complement pathways. Within the primary sequence of C4d, there is a reactive thioester group that binds covalently to nearby surfaces, thus labeling the locations of complement activation. This feature makes C4d a target for immunohistochemical staining aimed to aid the diagnosis of, among others, the antibody-mediated rejection of transplanted organs, membranous glomerulonephritis, bullous pemphigoid, or inflammatory myopathies. However, the credibility of C4d immunostaining is debatable, as a high background in surrounding tissues and body fluids and diffused patterns of deposits in target structures are experienced with some of the available anti-C4d antibodies. Herein, we present an improved version of a rabbit anti-C4d antibody, originally raised against the C-terminal linear neoepitope of this complement fragment. Minor cross-reactivity with C4b and native C4 proteins, measured by ELISAs, as well as relatively low concentrations necessary for obtaining a specific signal in immunohistochemical analyses of formalin-fixed paraffin-embedded material, makes the improved antibody superior to commercially available rabbit monoclonal anti-C4d antibody SP91 dedicated to ex vivo diagnostics, as demonstrated by the staining of a panel of kidney transplant biopsies.

Morphologic and immunophenotypic evaluation of liver allograft biopsies with contemporaneous serum DSA measurements.

BACKGROUND: Acute antibody mediated rejection is increasingly identified in liver allografts as a unique form of alloimmune injury associated with donor specific antibodies (DSA). This manifests pathologically as microvascular injury and C4d uptake. Despite the liver allograft's relative resistance to alloimmune injury, liver allografts are not impervious to cellular and antibody-mediated rejection. METHODS: In this blinded control study, we evaluated CD163 immunohistochemistry and applied the Banff 2016 criteria for diagnosis of acute AMR on a group of indication allograft liver biopsies from DSA positive patients and compared them to indication biopsies from DSA negative controls. RESULTS: Most DSA positive patients were females (75%, p = .027), and underwent transplantation for HCV infection. Significant histopathological predictors of serum DSA positivity were Banff H-score (p = .01), moderate to severe cholestasis (p = .03), and CD163 score > 2 (p = .029). Other morphologic features that showed a trend with DSA positivity include Banff portal C4d-score (p = .06), bile ductular reaction (p = .07), and central perivenulitis (p = .07). The odds of DSA sMFI ≥5000 was 12.5 times higher in those with a C4d score >1 than those with a C4d score ≤ 1 (p = .04). Incidence of definite for aAMR in the DSA positive cohort was 25% (n = 5), and 0% in the DSA negative cohort. A group of 5 DSA positive cases were not classifiable by the current scheme. CONCLUSION: Sinusoidal CD163, Banff H-score, and diffuse C4d are predictors of serum DSA, and facilitate recognition of histopathological features associated with serum DSA and tissue-antibody interaction.

Evidence for Alloimmune Sinusoidal Injury in De Novo Nodular Regenerative Hyperplasia After Liver Transplantation.

Posttransplant nodular regenerative hyperplasia (NRH) mostly remains unexplained. Microvascular injury due to antibody-mediated rejection (AMR) is suspected, but lack of donor specific antibody (DSA) testing makes it difficult to prove. Centered around a 1-year period of routine DSA testing, concomitant protocol, and indicated posttransplant liver biopsies (LB), recipients with NRH (n = 18) were compared with a matched control group (n = 36). All index, previous, and subsequent LB were reviewed. Both groups were similar in terms of demographics, timing of index LB, and DSA. In the index LB, the NRH group had higher sinusoidal C4d positivity (p = 0.029) and perisinusoidal fibrosis (p = 0.034), both independently associated with NRH (p = 0.038 and 0.050, respectively). Features of "possible" chronic AMR were detected in 28.5% of the NRH group without a known cause and 0% of the control group (p = 0.009). The NRH group had more preceding indicated LB with increased incidence of rejection and biliary obstruction pattern. In the follow-up histology, overall, sinusoidal and portal C4d positivity, sinusoidal microvasculitis, and perisinusoidal fibrosis were also higher (all p < 0.050). In conclusion, we provide evidence towards the hypothesis that some cases of posttransplant NRH are related to preceding active and persistent AMR. Large multicenter studies with protocol DSA testing are required to confirm.

Association between glomerular C4d deposition, proteinuria, and disease severity in children with IgA nephropathy.

BACKGROUND: C4d may be used as a marker to evaluate the condition and prognosis of adults with IgA nephropathy, but there have been few studies of children with IgA nephropathy. METHODS: C4d immunohistochemical staining was performed on samples from children with IgA nephropathy with C1q-negative immunofluorescence. The clinical and pathological treatment and prognostic characteristics of children in the C4d-positive and -negative groups were compared. RESULTS: A total of sixty-five children with IgA nephropathy were included in the study and were followed up for an average of 37 months. C4d was mainly deposited along the capillary loops. The urinary protein-to-creatinine ratio (UPCR) in the C4d-positive group was significantly higher than that in the C4d-negative group (3.97 vs. 0.81, P < 0.001), and the average integrated optical density value of each child was positively correlated with the UPCR (r = 0.441, P < 0.001). There was a significant difference in the proportions of children with mesangial hypercellularity (M1) (68.97% vs. 44.44%, P = 0.048) and segmental glomerulosclerosis (S1) (65.52% vs. 33.33%, P = 0.010) between the C4d-positive group and the C4d-negative group. The proportion of children who received immunosuppressants in the C4d-positive group was higher than that in the C4d-negative group (86.21% vs. 36.11%, P < 0.001). There was no significant difference in the proportion of children developing kidney failure between the two groups. CONCLUSION: C4d was found to be associated with proteinuria, segmental lesions, and immunosuppressant treatment. Activation of the lectin pathway may reflect the severity of clinical and pathological manifestations of IgA nephropathy in children. A higher resolution version of the Graphical abstract is available as Supplementary information.

Clinical relevance of glomerular C4d deposition in children with early IgA nephropathy or Henoch-Schönlein purpura nephropathy.

BACKGROUND: Both IgA nephropathy (IgAN) and Henoch-Schönlein purpura nephropathy (HSPN) are characterized by glomerular mesangial IgA deposition. Several large studies on adults have suggested that glomerular C4d deposition has prognostic value in IgAN. However, there are few relevant studies on the clinical value of C4d deposition in children with IgAN or HSPN. METHODS: We performed a retrospective cohort study in pediatric patients with IgAN or HSPN. Clinicopathological data were collected at the time of kidney biopsy. Kidney C4d deposition was analyzed by immunohistochemistry. The end point was defined as a ≥ 20% decrease in estimated glomerular filtration from baseline. RESULTS: We enrolled 75 children, including 36 children with IgAN and 39 with HSPN. The prevalence of C4d deposition was 36% (27/75). C4d deposition was more abundant in children with proteinuria ≥ 50 mg/kg/day (51.9% versus 20.8%, P = 0.006) or nephrotic syndrome (37.0% versus 10.4%, P = 0.006). Mesangial hypercellularity (hazard ratio [HR], 5.745, 95% confidence interval [CI], 1.670-19.761, P = 0.006) and IgM deposition (HR, 4.522, 95% CI, 1.321-15.478, P = 0.016) were associated with C4d deposition. After a median follow-up of 22 months, seven (19.4%) IgAN patients and one (2.6%) HSPN patient had decreased kidney function. In children with IgAN, positive C4d was associated with decreased kidney function (P = 0.047). CONCLUSION: Glomerular C4d deposition was associated with mesangial hypercellularity and glomerular IgM deposition in IgAN and HSPN. Glomerular C4d deposition may be a risk factor for eGFR decline in children with IgAN. A higher resolution version of the Graphical abstract is available as Supplementary information.

Patterns of Interferon γ Expression and C4d Deposition in Chronic Intervillositis of Unknown Etiology.

The pathogenesis of chronic intervillositis of unknown etiology (CIUE) may involve IFNγ overexpression. This study assesses the extent of IFNγ expression in CIUE by immunohistochemistry and compares it to spontaneous pregnancy losses. C4d deposition is also assessed to see whether IFNγ and C4d might represent separate diagnostic categories. Placenta from first to early second trimester with high grade CIUE (CHG; 17 cases) and low grade CIUE (CLG; 12 cases) is compared to euploid (SPLN; 18 cases), aneuploid spontaneous pregnancy losses (SPLA, 17 cases), normal placenta (NP, 13 cases). Protein level expression of IFNγ and C4d is assessed on whole tissue sections by immunohistochemistry. 35% of CHG and 42% of CLG show some level of IFNγ expression localized to the luminal surface of syncytiotrophoblast. 12% of SPLA and no SPLN or NP cases are IFNγ positive. C4d deposition is seen in 100% of CIUE, 88% of SPLA, 83% of SPLN, and 46% of NP samples. IFNγ overexpression occurs in approximately 40% of CIUE-related pregnancy losses. IFNγ expression restricted to a subgroup of CIUE implies that IFNγ may define a distinct disease process. The non-discriminatory pattern of C4d deposition suggests it is a non-specific phenomenon possibly related to placental damage.

C4d Staining Is Present in Normal Placentas From Pregnancies Prior to Pregnancy Loss Associated With Chronic Histiocytic Intervillositis and Is Reduced by Immunomodulatory Therapy in Subsequent Pregnancies.

INTRODUCTION: Chronic histiocytic intervillositis (CHI) is associated with adverse pregnancy outcomes and high recurrence risk. Recent studies suggest that CHI may represent a host-vs-graft rejection, and that C4d immunostain can be used as a marker for complement activation and antibody-mediated rejection in the CHI. MATERIALS AND METHODS: This retrospective cohort study focused on 5 fetal autopsy cases associated with CHI (5 index cases) from 5 women. We analyzed placentas from the index cases (fetal autopsy cases associated with CHI) and placentas from the women's previous and subsequent pregnancies. We assessed the presence and extent of CHI and C4d immunostaining in these placentas. We evaluated each available placenta and graded the severity of CHI as either <50% or ≥50%. Additionally, we conducted C4d immunostaining on one representative section from each placenta and graded the staining levels as follows: 0+ for staining <5%; 1+ for staining between 5% and <25%; 2+ for staining between 25% and <75%; and 3+ for staining ≥75%. RESULTS: Three of the 5 women had pregnancies prior to their index cases (fetal autopsy cases associated with CHI). Despite the absence of CHI in their initial pregnancies, the placentas displayed positive C4d staining with grades of 1+, 3+, and 3+, respectively. These results suggest the presence of complement activation and antibody-mediated rejection in placentas from their prior pregnancies without CHI. Three of the 5 women received immunomodulatory therapy after experiencing pregnancy losses associated with CHI. After treatment, 2 of these women had live births at 35 and 37 gestational weeks, respectively, while the third had a stillbirth at 25 gestational weeks. The severity of CHI and the degree of C4d staining in the placentas decreased in all 3 cases following immunomodulatory therapies. Specifically, the level of C4d staining decreased from 3+ to 2+, 2+ to 0+, and 3+ to 1+ in these 3 cases, respectively. DISCUSSION: In women with a history of recurrent pregnancy loss associated with CHI, C4d immunostaining was present in the placentas from their previous non-CHI pregnancies, suggesting activation of the classical complement pathway and antibody-mediated reaction in their prior non-CHI pregnancies before the development of CHI in subsequent pregnancies. Immunomodulatory therapy may improve pregnancy outcomes by reducing complement activation, as shown by the reduction of C4d immunopositivity in the placentas after immunomodulatory treatment. Although we believe that the study provides valuable insights, we acknowledge that there are limitations to the findings. Therefore, to further elucidate the pathogenesis of CHI, additional research efforts with a collaborative and multidisciplinary approach are necessary.

Complement activation negatively affects the platelet response to thrombopoietin receptor agonists in patients with immune thrombocytopenia: a prospective cohort study.

Increased platelet destruction is central in the pathogenesis of immune thrombocytopenia. However, impaired platelet production is also relevant and its significance underlies the rationale for treatment with thrombopoietin receptor agonists (TPO-RAs). Previous studies have associated enhanced complement activation with increased disease severity. Additionally, treatment refractoriness has been demonstrated to resolve by the administration of complement-targeted therapeutics in a subset of patients. The association between complement activation and the platelet response to TPO-RA therapy has previously not been investigated. In this study, blood samples from patients with immune thrombocytopenia (n = 15) were prospectively collected before and two, six and 12 weeks after the initiation of TPO-RA therapy. Plasma levels of complement degradation product C4d and soluble terminal complement complexes were assessed. Patients with significantly elevated baseline levels of terminal complement complexes exhibited more often an inadequate platelet response (p = .04), were exclusively subjected to rescue therapy with intravenous immunoglobulin (p = .02), and did not respond with a significant platelet count increase during the study period. C4d showed a significant (p = .01) ability to distinguish samples with significant terminal complement activation, implying engagement of the classical complement pathway. In conclusion, elevated levels of complement biomarkers were associated with a worse TPO-RA treatment response. Larger studies are needed to confirm these results. Biomarkers of complement activation may prove valuable as a prognostic tool to predict which patients that potentially could benefit from complement-inhibiting therapy in the future.

What is the context?Primary immune thrombocytopenia (ITP) is a potentially serious illness associated with an increased risk of bleeds. Manifestations range from confined skin bruising to life-threatening intracranial hemorrhages.It is an acquired immune disorder characterized by increased destruction and impaired production of platelets.Treatments aim at suppressing the destruction and supporting the production of platelets.Thrombopoietin receptor agonists (TPO-RA) are medically approved platelet growth factors that contribute to the generation of new platelets.The complement system is an evolutionary preserved part of innate immunity.Previous studies have indicated that complement activation may be an important contributor to disease and that the administration of complement-inhibiting therapy improves the platelet count in a subset of patients with primary ITP.What is new? The potential association between complement activation and a poor platelet response to TPO-RA therapy in primary ITP has not been previously studied.In fifteen patients with primary ITP starting TPO-RA therapy, we prospectively followed the platelet response and levels of complement biomarkers for 12 weeks.We showed that patients with high levels of complement biomarkers exhibited a worse treatment response during the study period.What is the impact?Our results suggest that levels of complement biomarkers may be valuable to predict which patients with treatment-refractory ITP that potentially could benefit from complement-inhibiting therapy in the futureLarger studies are needed to confirm our results.

Lactate and pyruvate levels correlation with lactate dehydrogenase gene expression and glucose consumption in Tamoxifen-resistant MCF-7 cells using capillary electrophoresis with contactless conductivity detection (CE-C4 D).

Breast cancer is the second leading cause of cancer death in women after lung cancer. The first-line treatment of metastatic breast cancer in premenopausal women relies on tamoxifen. The development of tamoxifen resistance is not fully understood. In this study, capillary electrophoresis with capacitively coupled contactless conductivity detector was developed to monitor the changes in lactate and pyruvate levels in supernatant media of three models of developed MCF-7 tamoxifen-resistant cells and correlate these metabolites changes with lactate dehydrogenase genes expression and glucose consumption. The electrophoretic separation was achieved under reversed electroosmotic flow conditions. The linear ranges were 0.15-5 and 0.01-1 mM with a correlation coefficient of 0.9966 and 0.9971 and the limits of detection were 0.01 and 0.02 µM for lactate and pyruvate, respectively. Inter- and intrarun accuracy were in the range of 96.88-105.94% with precision (CV, %) of ≤7.35%. The method was completely validated and the results were in agreement with those obtained using the lactate and glucose assay kits. The results revealed a significant increase in both lactate and pyruvate production in the three tamoxifen-resistant MCF-7 cells models compared to control cells. This increase was correlated with the increase of lactate dehydrogenase genes expression and the increase of glucose consumption.

The level of urinary C4d is associated with disease progression in IgA nephropathy with glomerular crescentic lesions: a cohort study.

BACKGROUND: Positive glomerular C4d staining, representative of lectin pathway activation, has been proven to be associated with unfavorable outcomes in immunoglobulin A nephropathy (IgAN). Our previous study suggested that urinary C4d correlated positively with an increase in crescents while the relationship between urinary C4d and disease severity and progression remains unelucidated. METHODS: In this study we enrolled 168 patients diagnosed with IgAN with varying proportions of crescent formation at the time of biopsy. An independent cohort of 107 IgAN patients was enrolled for validation. Kidney biopsy specimens were stained using immunohistochemistry. Urinary C4d levels at renal biopsy were measured by enzyme-linked immunosorbent assay. The primary endpoint was end-stage kidney disease (ESKD). RESULTS: Higher urinary C4d/creatinine levels were associated with a lower estimated glomerular filtration rate (eGFR); massive proteinuria; hypertension and severe Oxford M, E, T and C scores. After a median follow-up of 19 months (interquartile range 9-27), 53 (31.5%) participants reached ESKD. High urinary C4d/creatinine levels were independently and significantly associated with a risk of developing ESKD [hazard ratio per standard deviation increment of log-transformed C4d/creatinine 7.623 (95% confidence interval 4.117-14.113)]. CONCLUSIONS: The urinary C4d/creatinine level is a potential useful biomarker that was associated with disease severity and progression in patients with IgAN and crescents.

Relationship between immunoglobulin A1 lectin-binding specificities, mesangial C4d deposits and clinical phenotypes in immunoglobulin A nephropathy.

BACKGROUND: The reason why mesangial C4d deposits are detected in only certain biopsies of immunoglobulin A nephropathy (IGAN) remains unclear. We analyse the association between IgA glycosylation patterns, mesangial C4 deposition and clinical phenotypes in IgAN. METHODS: This cross-sectional study included 145 patients with idiopathic IgAN. We measured the serum levels of three different IgA1 lectin-binding specificities using enzyme-linked immunosorbent assays with and without treatment with neuraminidase and we analysed the relationship between these glycoforms, C4d mesangial deposits and clinical phenotypes. RESULTS: C4d-positive versus Cd4-negative patients had higher proteinuria [median 3.1 g/g (0.9-4.2) versus 1.8 (1-2.2); P = 0.000], haematuria [223 cells/µL (32-278) versus 99 (25-186); P = 0.000] and higher levels of IgA binding to neuraminidase untreated Helix aspersa (HA IgA1 neu-; 150.6 ± 52 U versus 96.2 ± 64.1; P = 0.000), neuraminidase untreated Helix pomatia (HPA IgA1 neu-; 0.34 ± 0.15 U versus 0.27 ± 0.13; P = 0.04), Triticum vulgaris (TV IgA1; 85.1 ± 31.7 U versus 42.2 ± 26.9; P = 0.000) and Canavalia ensiformis (ConA IgA1; 32.5 ± 18 U versus 16.7 ± 9.38; P = 0.000). The levels of HA IgA1 neu-, HPA IgA1 neu-, TV IgA1 and ConA IgA1 were all associated with the mesangial deposition of C4d, extracapillary proliferation and acute kidney injury. In receiver operating characteristics curves, HA IgA1 neu-, HPA IgA1 neu-, TV IgA1 and ConA IgA1 significantly discriminated between C4d-positive ad C4d-negative biopsies. In logistics models, TV IgA1 and ConA IgA1 were the only independent predictors of mesangial C4d deposits. CONCLUSIONS: In IgAN, the severity of the disease is associated with the level of IgA exposing N-acetyl-d-galactosamine, N-acetyl-d-glucosamine or mannose, whereas C4d deposits are only associated with elevated levels of IgA1 glycoforms exhibiting glycan residues with specificity for mannose and N-acetyl-d-glucosamine binding lectins.

Promoting weaning practices and growth of Egyptian infants by using communication for behavioral development approach.

BACKGROUND: Access to various affordable and nutritious foods is considered a challenging factor for households with limited resources affecting the proper weaning practices. In order to motivate communities to adhere to the right and proper weaning practices, the social aspect should be considered through close communication with the targeted communities. This study aimed to evaluate how impactful the use of the principles of Communication for Development (C4D) that respect parents' beliefs and their cultural norms is in improving the weaning practices and growth of infants in an Egyptian village. METHODS: An interventional three-phase study was conducted for three years. The intervention targeted 464 mothers of infants up to 2 years of age. C4D interventions encouraged each mother to provide her baby with nutritious and varied options through age-appropriate introduction and diversification of nutrient-rich complementary foods under the slogan " enjoy meals like a baby". The effectiveness of the approach was measured by five essential weaning practices: Introduction of solid, semi-solid, or soft foods, Minimum dietary diversity, minimum meal frequency, Minimum acceptable diet, and consumption of iron-rich foods. RESULTS: There was marked and significant improvement in the awareness and of the majority of the weaning practices' indicators as a result of the interventions. This was noticed for the timely introduction of complementary foods which increased from 36.7% to 82.0%, the minimum meal frequency indicator (3-5) which increased from 25.3% to 67.3%, iron-rich or fortified food (68.0% to 82%) as well as a regular checkup for baby health at the health unit (71.3%). Indicators that were improved but failed to achieve the target were the "Minimum Dietary Diversity" (reached 32%) and the minimum acceptable diet (reached 22.0%). A significant effect on linear growth especially for females is evidenced by the remarkable decrease in wasting (from 31.5% to 11.1%) and obesity (from 12.0% to 0%) associated with a considerable decrease in underweight (from 40% to 16.7%). CONCLUSION: Targeting caregivers through the C4D approach have succeeded in providing them with the support required for the provision of adequate nutrition for their infants that had significantly marked improvement in growth indices of their infants.