Raman spectroscopic differentiation of activated versus non-activated T lymphocytes: an in vitro study of an acute allograft rejection model.

Acute rejection (AR) remains a significant complication in renal transplant patients. Using serum creatinine for AR screening has proven problematic, and thus a noninvasive, highly sensitive and specific test is needed. T cells from human peripheral blood were analyzed using Raman spectroscopy. Fifty-one Mixed Lymphocyte Culture (MLC) activated T cells (ATC), 28 Mitomycin C inactivated T cells (ITC), and 35 resting T cells (RTC), were studied utilizing 785 and 514.5 nm wavelengths. Statistical analysis following subtraction of fluorescence used Student's t test to quantify peak ratio differences and discriminant function analysis (DFA), with three distinct sectors assigned for grouping purposes: Sector I, ITC; Sector II, ATC; Sector III, RTC. Differences between ATC and non-activated T cells (ITC and RTC) were found at 1182 and 1195 cm-1 peak positions for both wavelengths. Significant differences in peak ratios for 785 and 514.5 nm wavelengths existed between ATC and RTC (p=0.001 and p=0.006, respectively) and ATC and ITC (p=0.001 and p=0.001, respectively), with a trend in differences observed between ITC and RTC (p=0.07 and p=0.08, respectively). Analysis of the DFA-derived sector distribution for the 785 and 514.5 nm wavelengths revealed a sensitivity of 95.7% and 89.3%, respectively, and a specificity of 100% and 93.8%, respectively. This data suggests that Raman spectroscopy can detect significant differences between activated and nonactivated T cells based upon cell-surface receptor expression, thereby establishing unique signatures that can aid in the development of a noninvasive AR screening tool with high sensitivity and specificity.

Differentiation of alloreactive versus CD3/CD28 stimulated T-lymphocytes using Raman spectroscopy: a greater specificity for noninvasive acute renal allograft rejection detection.

Acute rejection (AR) remains problematic in renal transplantation. As a marker, serum creatinine is limited, warranting a more effective screening tool. Raman spectroscopy (RS) can detect T-cell activation with high sensitivity. In this study we explore its specificity. Seventy-five inactivated, 40 alloantigen-activated, and 75 CD3/CD28-activated T cells were analyzed using RS. CD3/CD28-activated peak magnitudes (PM) were 4.3% to 23.9% lower than inactivated PM at positions: 903, 1031, 1069, 1093, 1155, 1326, and 1449 cm(-1), with a difference in peak ratio (PR) observed at the 1182:1195 cm(-1) position (0.91 +/- 0.06 vs. 1.2 +/- 0.01, respectively: P = 0.006). Differences in CD3/CD28- and alloantigen-activated PM were observed at: 903, 1031, 1093, 1155, 1326, and 1449 cm(-1), with no PR differences at the 1182:1195 cm(-1) position (0.91 +/- 0.06 vs. 0.86 +/- 0.09: P = 0.8). Spectral signature separation of CD3/CD28-and alloantigen-activated groups was 100% specific and sensitive. We conclude that RS can differentiate T cells activated by different stimuli with high sensitivity and specificity.

Outcome predictors in African-American deceased-donor renal allograft recipients.

The relative importance of donor and recipient risk factors in predicting outcomes in African-American (AA) renal allograft recipients receiving contemporary immunosuppression, including early steroid withdrawal, has not been previously examined. We assessed the impact of 21 risk factors on five primary outcomes in 132 deceased-donor AA renal allograft recipients transplanted from July 2001 to August 2006 with follow-up 6-67 (mean 35 +/- 17) months by univariate and multivariate analysis. Thymoglobulin or basiliximab was given for induction, and mycophenolate mofetil with either tacrolimus or sirolimus (SRL) +/- prednisone for maintenance. Non-compliance accounted for 26% of graft loss (GL) and 19% of acute rejection (AR) episodes, and was more prevalent in patients who were HCV+ and those on prednisone. Delayed graft function remained a significant predictor of GL, but not via increased AR, and donor ethnicity emerged as an important predictor of patient death. De novo use of SRL resulted in increased AR, and only increased recipient age significantly predicted new-onset diabetes mellitus. Our preliminary results suggest the need for improvements in patient education, pre-transplant psychosocial assessment, and late post-transplant psychosocial support and can be utilized to help guide donor/recipient selection and tailor immunosuppressive management to optimize outcomes in this challenging group of patients.

Large adrenocortical carcinoma.

Adrenal cortical carcinomas (ACCs) are rare, highly malignant tumors that carry a poor prognosis. The large size and possibility of adherence to adjacent structures can make these tumors difficult to excise. We present a patient who underwent successful resection of a massive 19-cm, nonfunctional ACC, which encased the right kidney. The goal of this report is to enrich the growing body of knowledge concerning the presentation, evaluation, and surgical intervention of these rare cancers.

Preliminary experience with renal transplantation in HIV+ recipients: low acute rejection and infection rates.

BACKGROUND: Only four centers have reported their results with renal transplantation in human immunodeficiency virus (HIV)+ recipients on highly active antiretroviral therapy, and acute rejection (AR) rates have consistently ranged from 43% to 67%. METHODS: We examined the outcomes of eight adult HIV+ primary renal allograft recipients with median 15 (range 8-47) months follow-up with multiple other high-risk factors, including African American ethnicity, hepatitis C virus (HCV) positivity, long waiting times, prior sensitization, paucity of live donors, and delayed graft function. Our immunosuppressive protocol consisted of an anti-interleukin-2 receptor antibody for induction, and mycophenolate mofetil, cyclosporin A, and prednisone for maintenance. Initial and 3- to 6-month cyclosporin A trough level targets were 250 to 300 and 225 to 275 ng/mL, respectively, and mycophenolate mofetil dose was adjusted according to 2 to 4 week surveillance and subsequent as needed mycophenolic acid predose concentrations during the first 6 months. RESULTS: Patient and graft survival were 100% and 88%, respectively, with an AR rate of 13% and excellent renal function. No patients developed new-onset diabetes, opportunistic or other serious infections, malignancy, or progression of hepatitis C virus-related liver disease. Excellent suppression of HIV replication with maintenance of CD4 counts was noted in all cases. CONCLUSIONS: Our findings suggest that HIV+ patients on highly active antiretroviral therapy can undergo successful renal transplantation with a low incidence of both AR and AIDS-associated and non-AIDS associated infections, despite associated risk factors for poorer outcome. Our encouraging but preliminary results with this protocol will need to be verified in larger numbers of HIV+ renal allograft recipients with longer follow-up.

Raman spectroscopic modeling of early versus late T-lymphocyte activation via differential spectral detection of receptor expression.

The proven efficacy of renal transplantation has made it the definitive treatment for end-stage renal disease. Despite its wide acceptance, transplantation has been limited by organ shortages. In the face of this, preservation of allograft longevity is essential. The predominately T cell-driven process of acute rejection (AR) can lead to graft dysfunction and even graft loss. As a marker for AR screening, serum creatinine has a low sensitivity and specificity. This has warranted the development of more accurate screening/diagnostic tools such as Raman Spectroscopy (RS) which has been demonstrated in previous studies to accurately quantify T cell activation. In this study we further explore its application by modeling the dynamic process of cell surface receptor expression during T cell activation. 50 mitogen (Concanavalin A and pokeweed) activated T cells were stained with CD69, CD25, and CD71 monoclonal antibodies (mAbs) at 48 and 72 hour time points. In parallel, 50 activated T cells were analyzed using RS at these same time periods. At 4 8h there was high expression of the CD69 cell surface receptor detected via mAb staining with no appreciable binding of CD25/CD71 fluorescent tag. In conjunction, 48 hour RS-analyzed T cells demonstrated a significant peak difference at the 1585 cm(-1) position which represented a 63% (p=0.01) increase in peak magnitude when compared with the 72 hour samples. By contrast, the 72 hour data demonstrated an attenuation of the CD69 expression and increased CD25/CD71 expression. The corresponding RS analysis showed two significant peak differences at the 903 cm(-1) and 1449 cm(-1) positions that were not present at 48 h. These differences in Raman shifts resulted in a 40% (p=0.04) and a 59% (p=0.001) increase in peak magnitudes at these positions, respectively. This study serves to further validate RS as a screening modality capable of not only detecting T cells early in the activation process through the spectral signatures associated with CD69, but also quantifying the persistent expression of CD25 and CD71. This provides a foundation for the development of a system capable of the accurate assessment of acute and maintenance immunosuppression efficacy at the molecular level.

The role of endoscopic biliary sphincterotomy for the treatment of type 1 biliary dysfunction (papillary stenosis) with or without biliary stones.

BACKGROUND: This study assesses the safety and effectiveness of endoscopic biliary sphincterotomy (ES) in the treatment of papillary stenosis (PS) with and without biliary stones. METHODS: The records of all patients who had endoscopic retrograde cholangiopancreatography (2,689 patients) from January 1, 1991, to August 1, 2010, were reviewed. There were 117 patients with PS who had ES. RESULTS: All patients had biliary pain, a dilated common bile duct (CBD) with a maximum diameter of 10 to 25 mm, and elevated liver function tests. There were 46 patients who had prior cholecystectomy of whom 20 patients had CBD stones. The remaining 71 patients had no prior biliary surgery; there were no biliary stones in 14 patients. All patients were symptom free after ES with or without CBD stone retrieval. CONCLUSIONS: ES is the optimal treatment for PS in patients with or without biliary stones. ES eliminates pain, corrects CBD dilation, and restores LFTs to normal.

Does donor race still make a difference in deceased-donor African-American renal allograft recipients?

BACKGROUND: Prior studies have demonstrated that African-American (AA) donor kidneys are independently associated with an increased risk for graft loss. METHODS: We examined outcomes in comparable groups of AA deceased-donor (DD) kidney transplant patients receiving an AA donor (n=35) versus a Caucasian donor (C group; n=150) organ. RESULTS: There were no differences between AA and C groups in patient survival, new-onset diabetes, or BK nephropathy. The AA group demonstrated a significantly higher 6-month and overall incidence of acute rejection (AR), increased cytomegalovirus (CMV) infection, and decreased graft survival. Recurrent or de novo focal segmental glomerulosclerosis (FSGS) accounted for a significantly higher fraction of graft losses in the AA versus C group. CONCLUSIONS: AA DD renal allograft recipients have equivalent patient but decreased graft survival when transplanted with an AA versus C kidney using current immunosuppression. This may be the result of increased AR, CMV infection, and recurrence/development of FSGS.

Equivalent outcomes with primary and retransplantation in African-American deceased-donor renal allograft recipients.

BACKGROUND: Graft survival following renal retransplantation has been inferior to that following primary allografting, particularly in African Americans (AAs) receiving deceased-donor (DD) kidneys. METHODS: Among 166 AA DD renal allograft recipients transplanted from July 2001 through July 2007, we compared the outcomes of 26 (16%) receiving a second graft with those of 140 primary cases. All patients received either thymoglobulin (ATG) or an IL-2 receptor antagonist for induction, and were maintained on either tacrolimus or sirolimus + mycophenolate mofetil +/- prednisone. RESULTS: When compared with primary transplants, regrafts received kidneys from older donors, were younger, more sensitized, more likely to receive ATG and to be maintained on prednisone, received more doses of ATG, and were less likely diabetic. There was no difference between primary and retransplant groups in overall patient or graft survival; incidence of acute rejection, CMV infection, BK nephropathy, or new-onset diabetes mellitus; and serum creatinine at 1 year. CONCLUSION: AA renal allograft recipients can undergo a second DD transplant with intermediate-term outcomes comparable to that of a primary graft, despite the presence of multiple immunologic and non-immunologic high-risk factors, by extending the course of ATG induction and continuing prednisone therapy in the vast majority of cases.

Spontaneous gastric decompression of peripancreatic collection.

Peripancreatic collections related to acute pancreatitis can range from simple serous fluid to infected phlegmon. The latter can be associated with significant morbidity and mortality. A patient with striking abdominal computed tomography images of an infected phlegmonous peripancreatic collection was seen at our institution. The clinical picture was much different from what the images predicted. Endoscopic retrograde cholangiopancreatography revealed a fistulous connection between the collection and the stomach, also seen on retrospective abdominal computed tomography scan review, indicating spontaneous decompression, which cured the patient.

Intermediate-term outcomes of hepatitis C-positive compared with hepatitis C-negative deceased-donor renal allograft recipients.

BACKGROUND: Prior studies have yielded conflicting results concerning the impact of HCV on renal transplant outcomes. METHODS: We examined outcomes in comparable groups of predominantly African American hepatitis C virus (HCV)-positive (n = 34) and HCV-negative (n = 111) kidney transplant patients receiving contemporary immunosuppression. RESULTS: There was no difference in patient survival or acute rejection, but new-onset diabetes (NODM) was increased and graft survival decreased in the HCV-positive group, with increased graft loss secondary to noncompliance and Type I MPGN. The incidence of NODM among patients undergoing early corticosteroid withdrawal was 11% in both groups, while among those on prednisone, it was 47% in HCV-positive versus 25% in HCV-negative recipients. CONCLUSIONS: Deceased-donor HCV-positive renal allograft recipients have equivalent patient but decreased graft survival. Noncompliance and Type I MPGN play a role in producing this negative effect on graft outcome. Steroids may be required for HCV to exert its diabetogenicity in kidney transplant patients.

Carbon monoxide-induced status epilepticus in an adult.

There have been numerous reports documenting the occurrence of self-limiting seizures after carbon monoxide poisoning in the pediatric population. However, the literature has been less informative concerning the adult population. This report documents a 70-year-old female who was found to be in nonconvulsive status epilepticus after acute carbon monoxide poisoning with a carboxyhemoglobin level of 35%. To our knowledge, this is the first report of acute carbon monoxide poisoning inducing nonconvulsive status epilepticus in an adult. This report adds to the growing body of knowledge concerning acute carbon monoxide poisoning by further exploring the very complex symptomatology that presents in the adult population.

Abnormal cardiac wall motion and early matrix metalloproteinase activity.

Activation of matrix metalloproteinases (MMPs) in the heart is known to facilitate cardiac remodeling and progression to failure. We hypothesized that regional dyskinetic wall motion of the left ventricle would stimulate activation of MMPs. Abnormal wall motion at a target site on the anterior lateral wall of the left ventricle was induced by pacing atrial and ventricular sites of five open-chest anesthetized dogs. Changes in shortening at the left ventricular (LV) pacing site and at a remote site at the anterior base of the left ventricle were monitored with piezoelectric crystals. Simultaneous atrial and ventricular pacing resulted in abnormal motion at the LV pacing site, yielding early shortening and late systolic lengthening, whereas the shortening pattern at the remote site remained unaffected. Assessment of global myocardial MMP activity showed a sevenfold increase in substrate cleavage (P < 0.02) at the LV pacing site relative to the remote site. Gelatin zymography revealed increases in 92-kDa MMP-9 activity and 86-kDa MMP-9 activity at the LV pacing site relative to the remote site, whereas MMP-2 activity was unaffected. Abnormal wall motion was associated with increases in collagen degradation (approximately 2-fold; P < 0.03), plasmin activity (approximately 1.5-fold; P < 0.05), nitrotyrosine levels (approximately 20-fold; P = 0.05), and inflammatory infiltrate (approximately 2-fold; P < 0.02) relative to the remote site. Results indicate that regional dyskinesis induced by epicardial activation is sufficient to stimulate significant MMP activity in the heart, suggesting that abnormal wall motion is a stimulus for MMP activation.