Successful therapy with tonsillectomy for severe ISKDC grade VI Henoch–Schönlein purpura nephritis and persistent nephrotic syndrome

Henoch–Schönlein purpura (HSP) is a systemic disorder characterized by leukocytoclastic vasculitis involving the capillaries and the deposition of IgA immune complexes. Renal involvement is the principal cause of morbidity and mortality in children with HSP. We report here a 13-year-old girl with Henoch–Schönlein purpura nephritis (HSPN) of International Study of Kidney Disease in Children (ISKDC) grade VI and persistent nephrotic syndrome despite receiving conventional therapy, such as prednisolone, methylprednisolone and urokinase pulse therapy and plasmapheresis (PP). The patient was treated with tonsillectomy, which subsequently decreased proteinuria, induced the disappearance of microscopic hematuria, and improved renal pathological findings. A regimen of methylprednisolone and urokinase pulse therapy plus PP with tonsillectomy may be an effective and useful therapy for some children with severe HSPN children of ISKDC grade VI and persistent nephrotic syndrome.     

Peripheral blood IgA bearing cells in children with Henoch-Sch?nlein purpura nephritis and IgA nephropathy. Relationship between IgA bearing cells and clinico-pathological findings or T alpha 4 cells]

It has been reported that patients with Henoch-Sch?nlein purpura nephritis (HSPN) and IgA nephropathy (IgAN) showed an familial increase of IgA bearing peripheral blood lymphocyte. To elucidate the relationship between IgA bearing cells and clinico-pathological findings or T cell subsets, especially IgA specific helper T cells (T alpha 4 cells), 20 patients with HSPN and 33 patients with IgAN were studied. The results demonstrated as follows; 1) IgA bearing cells were significantly increased in patients with both HSPN and IgAN (p < 0.001). 2) The increase of IgA bearing cells was well correlated to the degree of patients' proteinuria and hematuria (p < 0.05), and also correlated with the severity of patients' renal pathological findings in both diseases. 3) With relation to the T cell subsets in patients with both diseases, only the CD4+ Leu8- cells (helper T cells) and T alpha 4 cells were significantly increased, in addition, positive correlation between IgA bearing cells and CD4+ Leu8- cells or T alpha 4 cells was observed. 4) The increase of IgA bearing cells seemed to be transient in HSPN, but remained elevated in IgAN. In conclusion, it was indicated that patients with HSPN and IgAN could have IgA related immunological abnormalities, which may be reflected in the increase of IgA bearing cells and T alpha 4 cells. It was also suggested that determination of IgA bearing cells could be a useful parameter which may be reflected in the disease activity of HSPN and IgAN.     

CD4+CD28null T cells are not alloreactive unless stimulated by interleukin‐15

Proinflammatory, cytotoxic CD4⁺CD28^null T cells can be substantially expanded in patients with end‐stage renal disease. These cells have been associated with the risk for rejection, but their alloreactive potential is unknown. CD4⁺CD28^null T cells were stimulated with HLA‐mismatched antigen presenting cells in the absence/presence of exogenous cytokines. Alloreactive potential was evaluated based on proliferation, degranulation, cytotoxicity, and cytokine production. Further, their suppressive capacity was assessed by measuring inhibition of proliferating alloreactive CD28⁺ T cells. CD4⁺CD28^null T cells contained alloreactive (CD137⁺) T cells but did not proliferate in response to allogeneic stimulation, unless interleukin (IL)‐15 was added. However, they could proliferate on stimulation with cytomegalovirus antigen without exogenous cytokines. IL‐15 increased the frequency of proliferating alloreactive CD4⁺CD28^null T cells to 30.5% without inducing CD28 expression (P < .05). After allogeneic stimulation together with IL‐15 and IL‐21, frequency of degranulating CD107a⁺CD4⁺CD28^null T cells increased significantly from 0.6% to 5.8% (P < .001). Granzyme B and perforin positivity remained similar, but production of interferon‐γ and tumor necrosis factor‐α increased by the combination of IL‐15 and IL‐21 (P < .001 and P < .05, respectively). Finally, CD4⁺CD28^null T cells did not show significant suppression. Thus, CD4⁺CD28^null T cells represent a population with absent alloreactivity unless IL‐15 is present.     

Role of β2 integrins in glomerular leucocytes in Henoch-Schoenlein purpura nephritis: an age-matched comparative study with immunoglobulin A nephropathy

SUMMARY: A comparative immunohistological study was performed for the glomerular deposition of complements (C1q and C3c), fibrin/fibrinogen‐related antigen (FRA), the expression of intercellular adhesion molecule‐1 (ICAM‐1), and the infiltration of leucocytes bearing β₂ integrins (leucocyte function associated antigen‐1 (LFA‐1), complement receptor 3 (CR3) and complement receptor 4 (CR4)) on renal biopsy specimens from 49 cases with Henoch‐Schoenlein purpura nephritis (HSPN), and 49 age‐matched cases with immunoglobulin A nephropathy (IgAN). the glomerular expression of ICAM‐1 was signifcantly correlated with the glomerular infiltration of leucocyte function associated antigen (LFA)‐1⁺ leucocytes in both diseases, and with that of CR3⁺ leucocytes in HSPN. the expression of ICAM‐1 was closely localized with the infiltration of LFA‐1⁺ leucocytes in the study with double immunostaining. the incidence and intensity of glomerular deposition of FRA were significantly higher in HSPN than in IgAN (P< 0.001), and those of C3c were significantly lower in HSPN than in IgAN (P< 0.001). the glomerular deposition of FRA was significantly correlated with the glomerular infiltration of CR4⁺ leucocytes in HSPN (P<0.05) but not in IgAN. In contrast, the glomerular deposition of C3c was significantly correlated with the glomerular infiltration of CR4⁺ leucocytes in IgAN (P<0.05), but not in HSPN. Studies with double immunostaining revealed a close association of CR4⁺ leucocytes with FRA deposition in HSPN and with C3c deposition in IgAN, respectively. the number of glomerular leucocytes bearing β₂ integrins was significantly correlated with urinary protein at the time of renal biopsy in both diseases. These results suggested the differential roles of β₂ integrins in the induction of glomerular injury in HSPN and IgAN. the ICAM‐1/LFA‐1 interaction may commonly be involved in the glomerular infiltration of leucocytes in both diseases. the ICAM‐1/CR3 interaction may be involved only in HSPN. Complement receptor 4 may function as a fibrin/fibrinogen receptor in HSPN, while CR4 may function as a complement receptor in IgAN.     

CD74 expression is increased in high‐grade,invasive urothelial carcinoma of the bladder

This study aimed to identify the clinicopathological features of bladder cancer patients with high CD74 expression, as milatuzumab humanized anti‐CD74 antibody is being evaluated in clinical trials for hematological malignancies. Expression of CD74 was examined in 342 urothelial carcinomas of the bladder, and two urothelial carcinoma cell lines by immunohistochemistry and western blotting, respectively. CD74 was overexpressed in 192 (56.1%) of the 342 cancer tissues, although it was not expressed in the cancer cell lines. CD74 staining was intense in tumor cells and inflammatory cells in the tumor stroma, but not in normal urothelium. CD74 expression was significantly associated with older age at diagnosis (≥68 years, P = 0.048), high World Health Organization grade (P = 0.019), advanced stages (P = 0.001) and non‐papillary growth pattern (P = 0.040). CD74 expression was also correlated with the absence of tumor‐infiltrating inflammatory cells (P < 0.001) and the presence of tumor‐associated inflammatory cells (P = 0.017). However, CD74 expression was not related to recurrence‐free and overall survivals in primary and subgroup analyses. In conclusion, urothelial bladder carcinomas with high CD74 expression are characterized by older age, high World Health Organization grade, non‐papillary growth and advanced stages.     

The study of mitoxantrone as a potential immunosuppressor in transgenic pig renal xenotransplantation in baboons: comparison with cyclophosphamide

Mounting evidence suggests that delayed xenograft rejection (DXR) of discordant xenografts has a strong humoral component. To explore the possibility of targeting this humoral response more efficiently, we performed a preliminary study in baboons immunized against pig blood cells using the immunosuppressor mitoxantrone (Mx). The results from this study showed that, in comparison with cyclophosphamide (CyP), Mx induced a long-lasting depletion of circulating B cells within 6 days of its administration and delayed secondary anti-Gal antibody (Ab) responses to pig blood cell immunizations. Given these results, we next evaluated Mx in an in vivo model of pig to baboon renal xenotransplantation. We performed a series of renal xenotransplantations in baboons using human CD55-CD59 transgenic donor pigs. In the first group of baboons (Mx group; n = 4) Mx was administered 6 days prior to the day of transplantation, the objective being to perform the xenotransplantation in a context where the recipient would have few remaining circulating B cells and thus have an impaired capacity to mount an Ab response to the xenograft. We compared this group to a second group of baboons treated with CyP starting 1 day prior to transplantation (CyP group; n = 2). All baboons receiving Mx or CyP received an additional immunosuppression of cyclosporin A, mycophenolate mofetil and steroids. No hyperacute rejection was observed in either group but all xenografts underwent DXR. Mx did not show superiority to CyP in terms of graft survival with a mean survival time of 8 +/- 2 days compared with 9 days for both CyP-treated baboons. Neither CyP nor Mx decreased serum levels of pre-existing anti-Gal Abs but levels of these Abs decreased dramatically within 1 day of transplantation, likely reflecting their immediate trapping within the xenograft. Interestingly however, in contrast to CyP, Mx inhibited the return of anti-Gal immunoglobulin M (IgM) to the circulation, even at the time of rejection. Nevertheless, strong intragraft deposits of IgM, IgG and the activated complement complex C5b-9 were observed in biopsies at rejection. Furthermore, despite the expected profound depletion of circulating B cells by Mx within 6 days of its administration, biopsies from both groups at rejection displayed a mild B cell infiltrate accompanied by a strong macrophage and intermediate T-cell infiltration, the latter tending to be more abundant in Mx-treated animals. Our data show that in this particular model of pig to baboon xenotransplantation and at the dose used, Mx was not superior to CyP in conferring protection against rejection, despite its capacity to profoundly deplete circulating B cells and to inhibit anti-Gal Ab responses to xenografts. DXR was thus possible without the return of anti-Gal Abs and may have been mediated by the early fixation of pre-existing Abs with secondary complement activation. However, although Mx was not more efficient than CyP in controlling DXR, its capacity to deplete B cells and delay Ab recovery may be beneficial in the context of Gal knockout organ transplantation where the induced Ab response is likely to take precedence over the preformed response.     

Siplizumab selectively depletes effector memory T cells and promotes a relative expansion of alloreactive regulatory T cells in vitro

Siplizumab, a humanized anti‐CD2 monoclonal antibody, has been used in conditioning regimens for hematopoietic cell transplantation and tolerance induction with combined kidney‐bone marrow transplantation. Siplizumab‐based tolerance induction regimens deplete T cells globally while enriching regulatory T cells (Tregs) early posttransplantation. Siplizumab inhibits allogeneic mixed‐lymphocyte reactions (MLRs) in vitro. We compared the impact of siplizumab on Tregs versus other T cell subsets in HLA‐mismatched allogeneic MLRs using PBMCs. Siplizumab predominantly reduced the percentage of CD4⁺ and CD8⁺ effector memory T cells, which express higher CD2 levels than naïve T cells or resting Tregs. Conversely, siplizumab enriched proliferating CD45RA^? FoxP3^HI cells in MLRs. FoxP3 expression was stable over time in siplizumab‐containing cultures, consistent with enrichment for bona fide Tregs. Consistently, high‐throughput TCRβ CDR3 sequencing of sorted unstimulated and proliferating T cells in MLRs revealed selective expansion of donor‐reactive Tregs along with depletion of donor‐reactive CD4⁺ effector/memory T cells in siplizumab‐containing MLRs. These results indicate that siplizumab may have immunomodulatory functions that may contribute to its success in tolerance‐inducing regimens. Our studies also confirm that naïve in addition to effector/memory T cells contribute to the allogeneic MLR and mandate further investigation of the impact of siplizumab on alloreactive naïve T cells.     

Clinical outcomes in children with Henoch-Schönlein purpura nephritis grade IIIa or IIIb

Henoch–Sch?nlein purpura (HSP) is one of the most common causes of systemic vasculitis in children. The incidence of HSP nephritis (HSPN) among HSP patients has been reported to be 15–62%. Even so, what constitutes severe HSPN is controversial. In the study reported here, we retrospectively reviewed the clinical features and prognosis of 101 children with HSPN, ISKDC grade IIIa/IIIb, from January 1992 to November 2008. Patients with isolated hematuria and/or proteinuria <50 mg/kg/day received triptolide alone, and those with nephrotic range proteinuria received a combination therapy of prednisone and triptolide. Nephrotic syndrome was the most common clinical manifestation (45.5%). There were no significant differences in the clinical features (χ² = 2.756, P = 0.252), the side effects related to treatment (χ² = 2.259, P = 0.894), prognosis between IIIa and IIIb (χ² = 3.013, P = 0.222), or prognosis in grade IIIa patients receiving triptolide alone or triptolide and prednisone (χ² = 1.207, P = 0.272) and grade IIIb patients (χ² = 1.158, P = 0.282). No significant difference in clinical manifestations and long-term prognosis of our HSPN patients with grade IIIa or grade IIIb were found, implying that our patients with International Study and Kidney Disease in Children (ISKDC) grade IIIb were not the most severe cases of HSPN. Our results may also suggest that treatment with steroid may not alter the clinical outcome of such grade IIIa or IIIb patients.     

Cut‐off values for serum matrix metalloproteinase‐9: Is there a threshold to predict renal involvement for Henoch–Schonlein purpura in children?

Aim: To clarify whether the level of matrix metalloproteinase‐9 (MMP‐9), tissue inhibitor matrix metalloproteinase‐1 (TIMP‐1) or the ratio of MMP‐9/TIMP‐1 was associated with the renal involvement in Henoch–Schonlein purpura (HSP); and to explore whether there existed early diagnostic measure for HSP nephritis (HSPN). Methods: Sixty‐six patients with HSPN, 68 patients with HSP and 60 healthy children (control group) were enrolled into our study. Serum and urine samples before treatment were collected for detection. Results: Compared with the HSP group and control group, serum MMP‐9, TIMP‐1 and ratio of MMP‐9/TIMP‐1 in the HSPN group were significantly higher (P < 0.05 and P < 0.01, respectively). Urine MMP‐9, TIMP‐1 and ratio of MMP‐9/TIMP‐1 in the HSPN group were obviously higher than those of the control group (P < 0.05) and the HSP group (P < 0.05). Receiver–operator curve (ROC) analysis was performed to obtain the area under the curve (AUC) and the AUC and its 95% confidence interval (CI) of serum MMP‐9 were 0.97 and 0.95–0.99, respectively. The optimal cut‐off point (sensitivity; specificity) of serum MMP‐9 for diagnosing HSPN was 179.79 mg/L (0.96; 0.88). Conclusion: Levels of MMP‐9, TIMP‐1 and ratio of MMP‐9/TIMP‐1 in serum and urine were remarkably high in the patients with HSPN, but the serum MMP‐9 was more sensitive. Serum MMP‐9 may be associated with the occurrence and development of renal involvement in HSPN and become an important indicator for early diagnosis of HSPN.     

Cyclooxygenase-2 promotes angiogenesis in pTa/T1 urothelial bladder carcinoma but does not predict recurrence

OBJECTIVE: To investigate the effect of cyclooxygenase-2 (COX-2) on microvessel density (MVD) and on the clinical prognosis in patients with non-muscle invasive urothelial carcinoma of the bladder, as COX-2 expression is significantly greater in epithelial tumours and there is increasing evidence that COX-2 might contribute to tumour neovascularization. PATIENTS AND METHODS: We assessed tumour samples from 110 patients undergoing transurethral resection for primary pTa/pT1 bladder carcinoma (pTa, 84; pT1, 26; grade 1, 22; grade 2, 81; grade 3, seven). Paraffin sections were assessed immunohistochemically using antibodies against COX-2, CD34 (endothelial cells) and CD105 (proliferating vessels). COX-2 expression was quantified by the number of stained cells (negative, +, ++) and the MVD calculated as vessels per field. RESULTS: Of the 110 tumours, 45 (41%) had no immunostaining for COX-2, 40 had faint staining with at least isolated positive cells (+) and 25 stained ++. COX-2 positive tumours had significantly greater vascularization for proliferating vessels. In COX-2 negative tumours the MVD was 22.1, identified by CD34 immunostaining, and 3.4 for proliferating vessels (CD105), whereas COX-2 positive tumours had a MVD of 18.3 (CD34), and of 5.8, respectively (CD105). Complete follow-up data were available in 91 patients; after a mean follow-up of 25 months, 18 (20%) had tumour recurrences. There was no significant difference in the recurrence rates or disease-free survival between COX-2-positive (19%, 25.6 months) or -negative patients (21%, 25.2 months). CONCLUSION: These results confirm the involvement of COX-2 in angiogenesis in bladder cancer, as COX-2 promoted blood vessel proliferation in the tumour zone, and indicate the usefulness of COX-2-inhibiting drugs in preventing and treating superficial bladder cancer.     

CMV‐specific T‐cell immunity,viral load,and clinical outcome in seropositive renal transplant recipients: a pilot study

Sund F, Lidehäll A‐K, Claesson K, Foss A, Tötterman TH, Korsgren O, Eriksson B‐M. CMV‐specific T‐cell immunity, viral load, and clinical outcome in seropositive renal transplant recipients: a pilot study.
Clin Transplant 2010: 24: 401–409. © 2009 Wiley Periodicals, Inc. Abstract: Background: Cytomegalovirus (CMV) infection is still the leading opportunistic infection following solid organ transplantation. The aim of this prospective study of renal transplant recipients was to evaluate the dynamics of CMV‐specific T‐cells, viral load, and clinical symptoms of CMV infection. Methods: Levels of tetramer‐selected CD8⁺ T‐cells (TetraCD8), CMV‐specific interferon‐γ producing CD8⁺ T‐cells (IFNγCD8), and CD4⁺ T‐cells (IFNγCD4), measured using major histocompatibility complex‐tetramer and cytokine flow cytometry techniques, and CMV DNA were monitored monthly in 17 CMV‐seropositive patients up to one yr (median 12 months, range 3–12) after transplantation and correlated to clinical outcome. Results: CMV DNAemia was detected in 94% of the patients, but only one patient developed CMV disease. CMV DNAemia >1 million copies/mL was seen in asymptomatic patients. CMV‐specific T‐cells decreased rapidly after transplantation. TetraCD8 and IFNγCD8 regenerated within three months, whereas IFNγCD4 recovery was impaired up to one yr after transplantation. The proportion of IFNγCD4 at two months post‐transplantation as compared with baseline, correlated strongly with the magnitude of the CMV DNAemia. Conclusions: Monitoring the reduction of IFNγCD4 compared with baseline during the first months after transplantation could be considered in predicting risk for high‐grade CMV DNAemia and in deciding strategic approaches for pre‐emptive and prophylactic therapy.     

Abnormal human chondrocyte morphology is related to increased levels of cell‐associated IL‐1β and disruption to pericellular collagen type VI

Early osteoarthritis (OA) is poorly understood, but abnormal chondrocyte morphology might be important. We studied IL‐1β and pericellular collagen type VI in morphologically normal and abnormal chondrocytes. In situ chondrocytes within explants from nondegenerate (grade 0/1) areas of human tibial plateaus (n = 21) were fluorescently labeled and visualized [2‐photon laser scanning microscopy (2PLSM)]. Normal chondrocytes exhibited a “smooth” membrane surface, whereas abnormal cells were defined as demonstrating ≥1 cytoplasmic process. Abnormal chondrocytes were further classified by number and average length of cytoplasmic processes/cell. IL‐1β or collagen type VI associated with single chondrocytes were visualized by fluorescence immuno‐histochemistry and confocal laser scanning microscopy (CLSM). Fluorescence was quantified as the number of positive voxels (i.e., 3D pixels with fluorescence above baseline)/cell. IL‐1β‐associated fluorescence increased between normal and all abnormal cells in the superficial (99.7 ± 29.8 [11 (72)] vs. 784 ± 382 [15 (132)]; p = 0.04, positive voxels/cell) and deep zones (66.5 ± 29.4 [9 (64)] vs. 795 ± 224 [9 (56)]; p = 0.006). There was a correlation (r² = 0.988) between the number of processes/cell (0–5) and IL‐1β, and an increase particularly with short processes (≤5 µm; p = 0.022). Collagen type VI coverage and thickness decreased (p < 0.001 and p = 0.005, respectively) with development of processes. Abnormal chondrocytes in macroscopically nondegenerate cartilage demonstrated a marked increase in IL‐1β and loss of pericellular type VI collagen, changes that could lead to cartilage degeneration. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1507–1514, 2010     

Allo‐HLA Cross‐Reactivities of Cytomegalovirus‐, Influenza‐, and Varicella Zoster Virus–Specific Memory T Cells Are Shared by Different Healthy Individuals

Virus‐specific T cells can recognize allogeneic HLA (allo‐HLA) through TCR cross‐reactivity. The allospecificity often differs by individual (private cross‐reactivity) but also can be shared by multiple individuals (public cross‐reactivity); however, only a few examples of the latter have been described. Because these could facilitate alloreactivity prediction in transplantation, we aimed to identify novel public cross‐reactivities of human virus‐specific CD8⁺ T cells directed against allo‐HLA by assessing their reactivity in mixed‐lymphocyte reactions. Further characterization was done by studying TCR usage with primer‐based DNA sequencing, cytokine production with ELISAs, and cytotoxicity with ⁵¹chromium‐release assays. We identified three novel public allo‐HLA cross‐reactivities of human virus‐specific CD8⁺ T cells. CMV B35/IPS CD8⁺ T cells cross‐reacted with HLA‐B51 and/or HLA‐B58/B57 (23% of tetramer‐positive individuals), FLU A2/GIL (influenza IMP[58‐66] HLA‐A*02:01/GILGFVFTL) CD8⁺ T cells with HLA‐B38 (90% of tetramer‐positive individuals), and VZV A2/ALW (varicella zoster virus IE62[593‐601] HLA‐A*02:01/ALWALPHAA) CD8⁺ T cells with HLA‐B55 (two unrelated individuals). Cross‐reactivity was tested against different cell types including endothelial and epithelial cells. All cross‐reactive T cells expressed a memory phenotype, emphasizing the importance for transplantation. We conclude that public allo‐HLA cross‐reactivity of virus‐specific memory T cells is not uncommon and may create novel opportunities for alloreactivity prediction and risk estimation in transplantation.     

Signaling through tumor necrosis receptor 2 induces stem cell marker in CD133+ regenerating tubular epithelial cells in acute cell‐mediated rejection of human renal allografts

Tumor necrosis factor receptor 2 (TNFR2) is strongly upregulated on renal tubular epithelial cells by acute cell‐mediated rejection (ACR. In human kidney organ culture, TNFR2 signaling both upregulates TNFR2 expression and promotes cell cycle entry of tubular epithelial cells. We find significantly more cells express CD133 mRNA and protein, a putative stem cell marker, in allograft biopsy samples with ACR compared to acute tubular injury without rejection or pretransplant “normal kidney” biopsy samples. Of CD133⁺ cells, ~85% are within injured tubules and ~15% are interstitial. Both populations express stem cell marker TRA‐1‐60 and TNFR2, but only tubular CD133⁺ cells express proximal tubular markers megalin and aquaporin‐1. TNFR2⁺CD133⁺ cells in tubules express proliferation marker phospho‐histone H3^S10 (pH3^S10). Tubular epithelial cells in normal kidney organ cultures respond to TNFR2 signaling by expressing CD133 mRNA and protein, stem cell marker TRA‐1‐60, and pH3^S10 within 3 hours of treatment. This rapid response time suggests that CD133⁺ cells in regenerating tubules of kidneys undergoing ACR represent proliferating tubular epithelial cells with TNFR2‐induced stem cell markers rather than expansion of resident stem cells. Infiltrating host mononuclear cells are a likely source of TNF as these changes are absent in acute tubular injury .     

Development and characterization of novel clinical grade neonatal porcine bone marrow‐derived mesenchymal stem cells

Due to recent advances in research on mesenchymal stem cells (MSCs), MSCs are expected to be used in various clinical applications. However, securing adequate cadaveric donors and safety of living donors are major issues. To solve such issues, we have examined to develop clinical grade neonatal porcine bone marrow‐derived MSCs (npBM‐MSCs). Clinical grade neonatal porcine bone marrow cells were collected, frozen, and sent to our laboratory by air. The npBM‐MSCs were isolated from thawed bone marrow cells, then frozen. The thawed npBM‐MSCs were examined for CD markers and differentiated into chondrocytes, osteocytes, and adipocytes. They were compared with human bone marrow‐derived MSCs (hBM‐MSCs) for growth rate and size. To assess the robustness of proliferation, we compared culture medium with or without gelatin. The npBM‐MSCs expressed positive MSC markers CD29, CD44, and CD90 and were differentiated into chondrocytes, osteocytes, and adipocytes. The doubling time of npBM‐MSCs was significantly shorter than that of hBM‐MSCs (17.3 ± 0.8 vs 62.0 ± 19.6 hours, P < 0.01). The size of npBM‐MSCs was also significantly smaller than that of hBM‐MSCs (13.1 ± 0.3 vs 17.5 ± 0.4 μm, P < 0.001). The npBM‐MSCs showed similar proliferation characters irrespective of with or without gelatin coating. The npBM‐MSCs secreted VEGF‐A, VEGF‐C, and TGF‐β1. We have established npBM‐MSCs which show super‐rapid growth, small size, and robust proliferation profile. The np‐MSCs might be able to solve the donor issues for MSC therapy.     

Treatment strategies for Henoch-Schönlein purpura nephritis by histological and clinical severity

The management of Henoch-Sch?nlein purpura nephritis (HSPN) is controversial. It has been revealed that some patients develop end-stage renal disease and aggressive treatment with drugs such as steroids is increasing, and some of them may be overzealous. At our institutes, our treatment decisions are based on the clinical and pathological severity of the case in an attempt to limit the indications for aggressive therapies such as steroids and immunosuppressive agents. Here, we retrospectively examined the efficacy of treatment for HSPN. Renal biopsy was performed in patients with nephrotic syndrome or persistent proteinuria for more than 3 months and patients were classified by treatment. Patients (n=31) with moderately severe HSPN (histological grade I-III and serum albumin [Alb] >2.5 g/dl) were treated with angiotensin-converting enzyme inhibitors and/or angiotensin receptor blockers. Patients (n=19) with HSPN exceeding grade III or Alb ≤ 2.5 g/dl received combination therapy comprising prednisolone, immunosuppressants, warfarin, and dipyridamole. All patients showed resolution of proteinuria without renal dysfunction during the observation period (3.76 ± 0.37 years). Our findings support those of some earlier reports that treatment strategies for HSPN should depend on the histological and clinical severity. Furthermore, aggressive therapies, particularly combination therapies, are unnecessary for moderate-severe HSPN.     

Intragraft proliferating T lymphocytes are associated with moderate acute pulmonary rejection

Acute allograft rejection is characterized by infiltration of the donor organ by host lymphoid cells, predominantly T lymphocytes. However, the site of proliferation and clonal expansion of alloreactive T lymphocytes is not well defined in man. A group of normal transbronchial biopsies (TBB, n=9) from clinically well lung transplant recipients was compared to TBB showing acute rejection (at least grade A2, n=9), using CD3- and Ki67-specific antibodies to double-label proliferating T lymphocytes. Few double-labeled lymphocytes were present in the normal biopsies (range, 0-3 cells). However, five of the rejection biopsies contained significant numbers of proliferating T lymphocytes (range, 19-47; Fisher's exact test; P=0.029). Furthermore, this positive group contained all three cases of grade A3 rejection in the study, as well as a case with persistent grade A2 rejection on follow-up biopsy. These data demonstrate that T lymphocytes do proliferate in transplanted human lungs; such proliferation is associated with more severe rejection.     

Antibody‐Mediated Rejection in Sensitized Nonhuman Primates: Modeling Human Biology

We have established a model of sensitization in nonhuman primates and tested two immunosuppressive regimens. Animals underwent fully mismatched skin transplantation, and donor‐specific antibody (DSA) response was monitored by flow cross‐match. Sensitized animals subsequently underwent kidney transplantation from their skin donor. Immunosuppression included tacrolimus, mycophenolate, and methylprednisolone. Three animals received basiliximab induction; compared with nonsensitized animals, they showed a shorter mean survival time (4.7 ± 3.1 vs. 187 ± 88 days). Six animals were treated with T cell depletion (anti‐CD4/CD8 mAbs), which prolonged survival (mean survival time 21.6 ± 19.0 days). All presensitized animals showed antibody‐mediated rejection (AMR). In two of three basiliximab‐injected animals, cellular rejection (ACR) was prominent. After T cell depletion, three of six monkeys experienced early acute rejection within 8 days with histological evidence of thrombotic microangiopathy and AMR. The remaining three monkeys survived 27–44 days, with mixed AMR and ACR. Most T cell–depleted animals experienced a rebound of DSA that correlated with deteriorating kidney function. We also found an increase in proliferating memory B cells (CD20⁺CD27⁺IgD^?Ki67⁺), lymph node follicular helper T cells (ICOS⁺PD‐1^hiCXCR5⁺CD4⁺), and germinal center (GC) response. Depletion controlled cell‐mediated rejection in sensitized nonhuman primates better than basiliximab, yet grafts were rejected with concomitant DSA rise. This model provides an opportunity to test novel desensitization strategies.     

Transplant Acceptance Following Anti‐CD4 Versus Anti‐CD40L Therapy: Evidence for Differential Maintenance of Graft‐Reactive T Cells

Inductive therapy with anti‐CD4 or anti‐CD40L monoclonal antibodies (mAb) leads to long‐term allograft acceptance but the immune parameters responsible for graft maintenance are not well understood. This study employed an adoptive transfer system in which cells from mice bearing long‐term cardiac allografts following inductive anti‐CD4 or anti‐CD40L therapy were transferred into severe combined immunodeficiency (SCID) allograft recipients. SCID recipients of cells from anti‐CD4‐treated mice (anti‐CD4 cells) did not reject allografts while those receiving cells from anti‐CD40L‐treated mice (anti‐CD40L cells) did reject allografts. Carboxyfluorescein succinimidyl ester (CFSE) labeling of transferred cells revealed that this difference was not associated with differential proliferative capacities of these cells in SCID recipients. Like cells from naïve mice, anti‐CD40L cells mounted a Th1 response following transfer while anti‐CD4 cells mounted a dominant Th2 response. Early (day 10) T‐cell priming was detectable in both groups of primary allograft recipients but persisted to day 30 only in recipients treated with anti‐CD4 mAb. Thus, anti‐CD40L therapy appears to result in graft‐reactive T cells with a naïve phenotype while anti‐CD4 therapy allows progression to an altered state of differentiation. Additional data herein support the notion that anti‐CD40L mAb targets activated, but not memory, cells for removal or functional silencing.     

CD154 Blockade Abrogates Allospecific Responses and Enhances CD4+ Regulatory T‐Cells in Mouse Orthotopic Lung Transplant

Acute cellular rejection (ACR) is a common and important clinical complication following lung transplantation. While there is a clinical need for the development of novel therapies to prevent ACR, the regulation of allospecific effector T‐cells in this process remains incompletely understood. Using the MHC‐mismatched mouse orthotopic lung transplant model, we investigated the short‐term role of anti‐CD154 mAb therapy alone on allograft pathology and alloimmune T‐cell effector responses. Untreated C57BL/6 recipients of BALB/c left lung allografts had high‐grade rejection and diminished CD4⁺: CD8⁺ graft ratios, marked by predominantly CD8⁺>CD4⁺ IFN‐γ⁺ allospecific effector responses at day 10, compared to isograft controls. Anti‐CD154 mAb therapy strikingly abrogated both CD8⁺ and CD4⁺ alloeffector responses and significantly increased lung allograft CD4⁺: CD8⁺ ratios. Examination of graft CD4⁺ T‐cells revealed significantly increased frequencies of CD4⁺CD25⁺Foxp3⁺ regulatory T‐cells in the lung allografts of anti‐CD154‐treated mice and was associated with significant attenuation of ACR compared to untreated controls. Together, these data show that CD154/CD40 costimulation blockade alone is sufficient to abrogate allospecific effector T‐cell responses and significantly shifts the lung allograft toward an environment predominated by CD4⁺ T regulatory cells in association with an attenuation of ACR.