A cluster of parvovirus B19 infections in renal transplant recipients: a prospective case series and review of the literature.

Up to 9% of renal transplant recipients have severe multifactorial erythropoietin-resistant anemia. Human parvovirus B19 (PVB19) infection can cause severe anemia and is likely underreported. Sparse information on epidemiology and management in this population exists. To address these issues, after our first index case, we modified our clinical practice to prospectively screen patients with persistent hemoglobin (Hb) <10 mg/dL for PVB19 infection after excluding common causes of anemia including erythropoietin resistance. Potentially infected patients were further evaluated by serology, qualitative polymerase chain reaction (quPCR) and bone marrow biopsy (BMB) for cytomegalovirus, Epstein-Barr virus, PVB19 and other etiologies. Over 3 months, 212 kidney recipients visited outpatient clinics. Of 52 recipients with anemia, 8 had an Hb <10 mg/dL with erythropoietin resistance and were screened for PVB19 infection. Three cases had PVB19 infection by quPCR and often-inconclusive serology/BMB results. Cases had immunosuppression reduced and received IVIG (0.5 gm/kg x 4 doses) with recovery from anemia, viral clearance in two cases and one recurrence. PVB19-mediated anemia occurred in up to three out of eight (38%) screened kidney recipients with Hb <10 mg/dL resistant to erythrypoietin. We recommend prospective risk stratification for this population, high indices of suspicion using at least qualitative techniques for diagnosis and treatment goal for viral eradication.

Screening a living kidney donor for polycystic kidney disease using heavily T2-weighted MRI.

Autosomal dominant polycystic kidney disease (ADPKD) accounts for approximately 8% of those awaiting renal transplantation. Living related kidney donors for these patients require screening for ADPKD, most commonly by ultrasonography. Ultrasound has a negative predictive value (NPV) of 100% in patients aged older than 30 years, but only 96% for donors aged 20 to 30 years. This case shows that heavily T2-weighted magnetic resonance imaging (HT2MRI) may be a more sensitive screening method for ADPKD in younger kidney donors. Despite a normal screening ultrasound result, a kidney donor with a family history of ADPKD was found to have renal cysts intraoperatively, and the transplantation was canceled. Afterward, the donor was imaged with HT2MRI. In addition, the mathematical relationship between sensitivity, specificity, and NPV for ADPKD screening tests was derived. After the canceled transplantation, a second ultrasound still could not identify renal cysts. However, HT2MRI showed multiple small ( approximately 3-mm) cysts in both kidneys and a 2.5-cm cyst on the right kidney. Mathematical analysis showed that the NPV of a screening test for ADPKD was most closely related to sensitivity and that only tests with 100% sensitivity would have a 100% NPV. We conclude that ultrasound is not a sufficiently sensitive screening test for ADPKD in younger living related renal donors. HT2MRI has improved sensitivity and may be the best screening test for ADPKD in this population.

Interleukin-2 and interferon-gamma double knockout mice reject heterotopic cardiac allografts.

BACKGROUND: Several studies suggest that MHC-mismatched allografts reject with a Th1 or Th2 immune response, but these models all have low-level expression of the Th1 cytokines interleukin (IL)-2 and interferon-gamma (IFN-gamma). METHODS: We interbred mice with single targeted gene disruptions for IL-2 and IFN-gamma to establish IL-2 + IFN-gamma double knockout (DKO) mice. Heterotopic cardiac allografts from DBA/2j (H2d) donors were transplanted WT, IL-2 knockout (KO), IFN-gamma KO, and DKO recipients (C57BL/6x129; H2b). Cytokine transcripts from allografts and DKO splenocytes were analyzed by reverse transcription polymerase chain reaction. RESULTS: DKO mice had a cytokine profile and IgG1/ IgG2a isotype ratio characteristic of Th2 deviation. DKO recipients rejected heterotopic cardiac allografts faster than IL-2 KO mice, but significantly slower than WT and IFN-gamma KO mice (P<0.01). Analysis of the rejecting DKO recipients showed intragraft Th2 cytokine expression. CONCLUSION: The combined absence of IL-2 and IFN-gamma in the setting of Th2 deviation does not prevent allograft rejection.

Combined costimulation blockade plus rapamycin but not cyclosporine produces permanent engraftment.

BACKGROUND: Combined treatment of allograft recipients with anti-CD40 ligand and CTLA-4Ig (costimulation blockade) is a powerful promising albeit not consistently tolerizing therapy. It would be desirable to use an effective conventional immunosuppressive regimen in low doses or for a short course as an adjunct; however, cyclosporine treatment drastically blunts the ability of costimulation blockade to produce long-term engraftment. METHODS: Short courses of cyclosporine or rapamycin were compared as adjuncts to costimulation blockade in the murine BALB/c to C3H/He heterotopic cardiac allograft model. RESULTS: Although cyclosporine therapy blocked the capacity of costimulation blockade to produce permanent engraftment, combined rapamycin and costimulation blockade treatment produced permanent engraftment. CONCLUSION: A theoretical basis for the differing effects of cyclosporine and rapamycin upon the outcome of costimulation blockade is forwarded. Combined use of costimulation blockade and rapamycin may provide a means to bring costimulation blockade into the clinic.

On histocompatibility barriers, Th1 to Th2 immune deviation, and the nature of the allograft responses.

In the present study, we have sought to determine the basis for the frequent failure of Th1 to Th2 immune deviation to blunt the severity of allograft rejection, as such immune deviation has proven highly effective in the treatment of several T cell-dependent autoimmune states. Our study demonstrates that treating islet allograft recipient mice with anti-IL-12 mAb is highly effective in producing Th1 to Th2 immune deviation in several model systems (i.e., fully MHC, partially MHC, or multiple minor Ag barriers). Nevertheless, anti-IL-12 failed to prolong the engraftment of fully MHC-mismatched islet allografts. However, anti-IL-12-treated recipients carrying MHC-matched but multiple minor Ag-mismatched allografts experienced prolonged engraftment; allograft tolerance was frequently achieved in the DBA/2J (H-2d) to BALB/c (H-2d) strain combination. In another model, in which the host response was dominated by CD4+ T cells responding to donor allopeptides presented upon host APCs in the context of self MHC class II molecules, anti-IL-12 treatment proved to be extremely potent. Thus, Th1 to Th2 immune deviation produces prolonged engraftment as compared with recipients of MHC-mismatched allografts when rejection is dependent upon indirectly presented allogeneic peptides and a reduced mass of responding alloreactive T cells.

Differential expression of T-cell growth factors in rejecting murine islet and human renal allografts: conspicuous absence of interleukin (IL)-9 despite expression of IL-2, IL-4, IL-7, and IL-15.

BACKGROUND: Interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15, all T-cell growth factors (TCGFs), utilize the common IL-2 receptor gammac chain as a critical signaling component in their receptor complexes. We have bred IL-2-/- and IL-4-/- double knockout (DKO) mice and showed vigorous islet allograft rejection by DKO hosts. The identity of TCGFs that support the IL-2- and IL-4-independent allograft rejection is unclear. METHODS: We analyzed IL-9 gene expression in rejecting islet allografts in wild-type and in DKO mice, as well as in human renal transplant biopsy specimens, by reverse transcriptase polymerase chain reaction and compared the expression of IL-9 with that of other TCGFs. RESULTS: IL-9 gene expression was not detected in rejecting murine islet allografts in either wild-type or DKO recipient mice despite robust expression of other TCGFs, including IL-7 and IL-15. IL-9 transcripts were also not expressed in any of the human renal transplant biopsies obtained 4 to 251 days after transplantation, regardless of the presence or absence of histological evidence of rejection. Despite expression of IL-9 by DKO splenic cells upon in vitro mitogenic stimulation, IL-9 alone was unable to stimulate the proliferation of concanavalin A-activated splenic leukocytes harvested from DKO mice. CONCLUSION: IL-9 is conspicuously absent despite vigorous expression of IL-2, IL-4, IL-7, and IL-15 genes during acute allograft rejection.

IL-2 and IL-4 double knockout mice reject islet allografts: a role for novel T cell growth factors in allograft rejection.

T cell growth factors (TCGFs) play a critical role in allograft rejection by promoting the activation and proliferation of alloreactive T cells. To determine whether IL-2 and IL-4 are of quintessential importance in allograft rejection and to identify possible alternative TCGFs, we have bred IL-2(-/-) and IL-4(-/-) double knockout (DKO) mice and studied islet allograft rejection using the DKO mice as allograft recipients. Although mononuclear leukocytes from DKO mice did not mount a proliferative response in vitro in response to anti-CD3 stimulation, crude islet allografts were vigorously rejected by DKO mice (mean survival time 17 +/- 7, n = 8) as compared with wild-type controls (mean survival time 13 +/- 4, n = 7). Treatment of DKO mice with anti-CD3 or rapamycin markedly prolonged the islet allograft survival. An analysis of intragraft cytokine gene transcripts showed robust expression of IL-7 and IL-15. In contrast, intragraft IL-9 gene transcripts were not detected in either wild-type or DKO mice. Provision of exogenous IL-2, IL-4, IL-7, or IL-15, but not IL-9, supports the proliferation of anti-CD3 activated DKO splenic leukocytes in vitro. Blocking the common gamma c of IL-2 receptor, a shared essential signaling component by receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, prolonged the survival of islet allografts in DKO mice. Hence, a T cell dependent allograft rejection enabled by rapamycin-sensitive signals or signals mediated by binding of the gamma c chain occurs in the absence of both IL-2 and IL-4. Non-T cell-derived TCGFs, especially IL-7 and IL-15, may play an active role in supporting allograft rejection.

Mechanical perturbation of webbed edges in 3T3 cells.

We have previously described actin edge-bundles (AEBs) as cables of microfilaments lining the webbed edges of 3T3 cells (Zand and Albrecht-Buehler: Cell Motil. Cytoskeleton 13:195-211, 1989). We have suggested that AEBs, along with their cell-substratum adhesions, resist cortical tension and prevent the collapse of cytoplasm towards the nucleus. In this paper, we report several stages of AEB disassembly and re-formation induced by the following micro-manipulations: (1) Scoring of the webbed edge of a 3T3 cells with a micro-needle. As a result the sides of the score retracted and the severed AEB appeared to disassemble down to its terminal adhesion points. The retraction stopped after 20-40 seconds and the cells formed a webbed edge with large curvature. Over a period of 20-80 minutes, the new web decreased in length and depth, until it regained its approximate original shape. (2) Bending of cell processes at acute angles. As a result the processes moved until they projected at right angles to the side of the cell and formed new webs gradually expanded their area. In both cases, the nascent webs were lined by actin edge-bundles.

What structures, besides adhesions, prevent spread cells from rounding up?

The outline of cells in sparse cultures consists predominantly of concave and convex segments; straight segments are rare and ephemeral. The convex segments are areas of active cell expansion. The concave segments are stationary and web-shaped, similar in profile to the cables of a suspension bridge. In 3T3 fibroblasts, we have found a single microfilament bundle following the outline of every webbed edge and have called it the actin edge-bundle (AEB). While the AEB is composed predominantly of actin, alpha-actinin and myosin are also present. In contrast to normal stress fibers, AEBs are more resistant to several treatments that depolymerize F-actin. Once an AEB disassembles, however, the webbed edge collapses and retracts, suggesting that the actin edge-bundle is a specialized cytoskeletal structure that supports the webbed edges of interphase 3T3 fibroblasts. The stability of AEBs is independent of microtubules. We suggest that the microfilament bundles that frequently line the lateral contacts between epithelial cells in vivo may be related to the actin edge-bundle.

Long-term observation of cultured cells by interference-reflection microscopy: near-infrared illumination and Y-contrast image processing.

Interference-reflection microscopy (IRM) is the only method presently available with which to visualize cell-substratum adhesions in living tissue culture cells continuously for long periods of time without the use of fluorescent markers (Curtis: J. Cell Biol. 20:199-215, 1964; Izzard and Lochner: J. Cell Sci. 21:129-159, 1976). This method utilizes approximately 1% of the incident illumination to produce the IRM image (Verschueren: J. Cell Sci. 75:279-301, 1985) and so far has required the use of high-intensity light sources in the visible spectral range (400-800 nm). Unfortunately, visible light of this intensity and spectral range induces marked changes in the behavior and morphology of motile fibroblasts, including cessation of locomotion. In contrast, the present paper reports that continuous observations of live cells in IRM for periods of up to 8 hours are possible if the illuminating light is in the red to near-infrared range (650-950 nm) and without any observable change in normal cell morphology or behavior. In addition, we describe how the technique of Y-contrast image processing can be applied to IRM images to create a three-dimensional image of the ventral cell surface topography.

Fatigue failure of cortical bone screws.

An experimental study of the fatigue life of cortical bone screws under conditions which stimulated in vivo usage was performed. The two most important factors influencing fatigue life were axial screw tension (the force normal of the plate to bone) and the cyclic shearing load. All screws failed at the root of the thread in the interface between the plate and the bone. A modified screw design effectively resisted fatigue under the described experimental conditions.