Portal hypertension as a result of the incomplete surgically treated advanced alveolar echinococcosis: a case description.

BACKGROUND: Infection of Echinococcus multilocularis causes in humans the alveolar echinococcosis. Although the infection has world-wide distribution it is rarely detected. Diagnosis of alveococcosis is difficult because of not typical clinical picture and irregular results of radiological examinations suggesting neoplasmatic process which begins in the liver tissue or in the biliary tracts. The parasitic growth is slow, so the illness is quite often established in late invasion period. Treatment of long-lasting and late diagnosed infection is difficult and requires cooperation of parasitologists together with surgeons to avoid life-threatening organ dysfunction. CASE PRESENTATION: We describe a young male patient, diagnosed, according to the radiological, immunological and histological examination results, infection of Echinococcus multilocularis, who was treated with not radical resection of pathologic mass together with persistent albendazole intake. The right hepatectomy was performed. In addition, visible cysts were removed from the left lobe of the liver in nonanatomical resection and suspicious calcified lesions in hepatoduodenal ligament were also removed. After the operation portal hypertension, with splenomegaly and symptoms of the liver cirrhosis occurred (thrombocytopenia, collateral venous circulation, first degree varices oesophagii). The portal hypertension probably could be a result of incomplete surgery due to extended parasitic infection and liver anathomical changes due to performed procedures, because the portal hypertension and it's further complications had not been observed before the operation. CONCLUSIONS: Echinococcus multilocularis should be taken under consideration in differential diagnosis of irregular lesions within the liver. Lon-lasting invasion could be responsible for the irreversible secondary liver changes such as cirrhosis and portal hypertension. The surgery treatment (treatment of choice) is difficult and it's results depends on the invasion period the patient is operated on. After the surgery the patient requires careful follow - up, to detect early complications.

"Extracorporeal Membrane Oxygenation for Greater Poland" Program: How to Save Lives and Develop Organ Donation?

The "ECMO for Greater Poland" program takes full advantage of the extracorporeal membrane oxygenation (ECMO) perfusion therapy opportunities to promote the health of the 3.5 million inhabitants in the region. The main implementation areas are treatment of patients with hypothermia; severe reversible respiratory failure (RRF); critical states resulting in heart failure, that is, cardiac arrest, cardiogenic shock, or acute intoxication; and promotion of the donor after circulatory death (DCD) strategy in selected organ donor cases, after unsuccessful life-saving treatment, to achieve organ recovery. This organizational model is complex and expensive, so we used advanced high-fidelity medical simulation tests to prepare for real-life experience. Over the course of 4 months we performed scenarios including "ECMO for DCD," "ECMO for extended cardiopulmonary resuscitation," "ECMO for RRF," and "ECMO in hypothermia." Soon after these simulations, Maastricht category II DCD procedures were performed involving real patients and resulting in 2 successful double kidney transplantations for the first time in Poland. One month later we treated 2 hypothermia patients (7 adult patients with heart failure and 5 patients with reversible respiratory failure) with ECMO for the first time in the region. Fortunately, we have discovered an important new role of medical simulation. It can be used not only for skills testing but also as a tool to create non-existing procedures and unavailable algorithms. The result of these program activities will promote the care and treatment of patients in critical condition with ECMO therapy as well as increase the potential organ pool from DCDs in the Greater Poland region of Poland.

The relationship between epileptiform discharges and background activity in the visual analysis of electroencephalographic examinations in dogs with seizures of different etiologies.

Electroencephalographic (EEG) recordings in 125 outpatient dogs with various epileptiform encephalopathies were acquired under medetomidine sedation using subdermal wire electrodes. The features of canine EEG (background activity [BGA] and epileptiform discharges [EDs]) were assessed, described and compared. The dogs included neurologically healthy controls (N, n = 19), dogs with portosystemic shunt (PSS, n = 9), dogs with intracranial pathologies (IP, n = 27) and dogs with idiopathic epilepsy (IE, n = 70). A visual EEG analysis revealed significantly more pronounced high voltage, low-frequency BGA in the PSS and IP groups in comparison to the N and IE groups (PSS vs. N, PSS vs. IE P <0.0001; IP vs. N, IP vs. IE P = 0.043). At least one ED in the recording was found in 47.37% (n = 9/19) of the individuals in the N group, 88.9% (n = 8/9) of the dogs in the PSS group, 77.78% (n = 21/27) of the dogs in the IP group and 61.43% (n = 43/70) of the dogs in the IE group. The presence of bilateral symmetric triphasic (BST) waves was significantly higher in the PSS group than in the remaining groups. There was a strong prevalence of spike-waves in dogs with idiopathic epilepsy and of BST waves in dogs with portosystemic shunt. None of the dogs in group N had spike-waves or BST activity. EDs were observed more frequently in high and very high voltage, low frequency BGA than in low voltage, high frequency BGA.

The expression profile of angiogenic genes in critical limb ischemia popliteal arteries.

Critical limb ischemia (CLI) represents the most severe form of peripheral arterial disease (PAD) and is the leading cause of non-traumatic amputations in western populations. In recent years, therapeutic angiogenesis has been considered to be a potential treatment option for CLI patients, however the molecular mechanism of ischemia-induced vascularization is still not fully understood. The identification of genetic factors underlying vascular responses to ischemia will improve our understanding of the biological causes of the disease and enhance personalized therapies in the future. In this work, we determined, for the first time, the expression profile of angiogenesis-related genes utilizing unique human material: the popliteal arteries retrieved during lower limb amputation from patients with CLI. Using custom-designed TaqMan Low-Density Array (TLDA) cards we investigated the mRNA level of 90 genes on CLI samples compared to healthy donors. We identified three significantly up-regulated genes in CLI group: matrix metalloproteinase 9 (MMP-9), VE-cadherin (CDH5) and integrin alpha 4 (ITGA4). However, among all investigated genes, only lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) was significantly reduced. In order to verify whether hypoxic conditions occur in popliteal arteries of CLI patients, we validated the transcription level of selected proangiogenic genes by real-time PCR on a larger number of samples. These results showed that the expression of key genes involved in angiogenesis, such as MMP9, HGF, HIF1A, VEGF-A and FLT1 were elevated in patients with CLI. Moreover, the study revealed that the expression of VEGF-A and FLT1 was associated with activation of HIF1A transcription. In conclusion, our data revealed the alteration in the mRNA level of genes involved in matrix remodelling, cell-cell adhesion as well as endothelial cell migration and proliferation in human popliteal arteries.

Identification of a molecular defect in a stillborn fetus with perinatal lethal hypophosphatasia using a disease-associated genome sequencing approach.

Lethal skeletal disorders represent a heterogeneous and clinically variable group of genetic conditions, usually difficult to diagnose without post-mortem radiological assessment. Here we report on a stillborn patient delivered at 22 weeks of gestation who presented with severe skeletal symptoms comprising limb shortening and intrauterine fractures detected upon prenatal ultrasound and autopsy examination. Since post-mortem X-ray was refused and no phenotypic diagnosis could be attempted, we performed next-generation sequencing (NGS) of 2741 genes associated with all known Mendelian disorders. With this strategy, we were able to demonstrate the diagnosis at a molecular level, which turned out to be perinatal lethal hypophosphatasia (HPP). This severe form of HPP represents an inborn defect of ossification often resulting in stillbirth or postnatal death. The NGS panel revealed compound heterozygous ALPL missense mutations: c.1283G>C(p.Arg428Pro) and c.1363G>A(p.Gly455Ser). Mutations detected in our case, although previously described in other patients, have not been reported to co-occur in a single individual. The diagnosis established in our index using the NGS-based approach could have been successfully reached by standard radiography. Thus, our report points to the importance of X-ray examination in stillborn cases and highlights the emerging role of NGS strategies in the diagnostic process of prenatally manifesting skeletal disorders.

Bacterial Infections in Renal Transplant Recipients.

BACKGROUND: Patients after kidney transplantation are highly susceptible to infections owing to immunosuppression as well as other risk factors--extended hospitalization, Foley catheterization, and double J catheter insertion among others. Bacterial infections, especially shortly after operation, are a major threat to the graft function. The aims of this study were to identify risk factors of bacterial infections after kidney transplantation and to determine the impact of those infections on the subsequent renal function. METHODS: One hundred twenty patients who underwent kidney transplantation in 2013 and 2014 were examined in our study for possible risk factors of bacterial infections and for possible outcome of such infections on their future condition. RESULTS: Among 120 patients under observation, 50 (41.7%) had early infectious complications (during hospitalization), 41 (82%) of which were urinary tract infections (UTI). The second most common infectious complication was infection of the surgical wound. Statistically significant results were obtained only for patient's age and duration of hospital stay (P = .001 and P = .000004, respectively). Bacterial infection resulted in longer hospital stay, higher reoperation risk, and lower creatinine clearance in the 14 days after transplantation (P = .000004, P = .0142, and P = .0455, respectively). CONCLUSION: Bacterial infections influence mainly the short-term condition of kidney transplant recipients: extended hospital stay, decreased early creatinine clearance, and enhanced risk of reoperation. The most common risk factors, such as Foley catheterization, double J catheter insertion, and diabetes, were not significant in our observation.

Is multiresistant Klebsiella pneumoniae New Delhi metallo-beta-lactamase (NDM-1) a new threat for kidney transplant recipients?

Urinary tract infections (UTIs) are the most frequent infections among kidney transplant (KT) patients. This case documents the emergence of New Delhi metallo-beta-lactamase (NDM-1) Klebsiella pneumonia--a factor of recurrent post-KT UTI, leading to graft loss. Spreading globally, and multidrug resistant, NDM-1 may become a great threat to transplant patients all over the world.

Urologic de novo malignancies after kidney transplantation: a single center experience.

INTRODUCTION: Urologic cancers are the second or third most common malignancies in renal transplant (RT) recipients. This study sought to determine the incidence of and identify possible risk factors for urologic malignancies among patients who underwent transplantation at our center. METHODS: This retrospective, single-center cohort included 836 patients who underwent transplantation from 1994 to 2011 who remained under our care. A review of their medical records revealed 63 subjects with de novo cancer, including 21 with urologic malignancies (2.5%). We analyzed demographic and clinical data of cancer versus noncancer patients with differences considered to be significant at P < .05. RESULTS: The urologic malignancies included renal cell carcinoma (n = 13), prostate cancer (n = 5), and bladder transitional cell carcinoma (n = 3). The mean follow-up time was 10 ± 3.9 years. The mean age at diagnosis was 54 ± 7.4 years and the mean time from transplantation was 4 ± 3.3 years. The mortality rate among group was 19.0%. The analysis did not show significant differences in demographic or clinical characteristics between the groups, except for the prevalence of male gender and smoking status among the cancer cohort. No significant differences were observed for other suspected risk factors, including immunosuppressive protocols, time of pretransplantation dialysis, and age. CONCLUSIONS: The development of urologic malignancies is an early event, frequently observed within 4-5 years after transplantation. Therefore, this period should be considered for routine urologic cancer screening.

Skin cancer following kidney transplantation: a single-center experience.

One of the major problems associated with prolonged immunosuppression is a high occurrence of skin malignancies among kidney recipients. Studies have shown that nonmelanoma skin cancer is the most frequently occurring tumor after organ transplantation. The aim of this study was to determine the incidence of and identify possible risk factors for skin malignancies among a population of kidney recipients. This retrospective, single-center cohort comprised 1672 patients transplanted from 1994 to 2011. Only patients with a confirmed diagnosis of skin cancer were selected for medical records review. Among 836 kidney transplant recipients remaining under our care since 1994, skin malignancies were diagnosed in 16 patients (1.9%). The histological diagnoses included squamous cell carcinoma (n=8; 50.0%); basal cell carcinoma (n=6; 37.5%) or malignant melanoma (n=2; 12.5%). The slightly lower incidence of skin malignancies noted in our study compared with other reports might result from differences in the length of follow-up. Some patients diagnosed with skin cancer were treated in local dermatology clinics. Also, a lower exposure to the sun characteristic for the latitude and differences in immunosuppressive therapies could be partially responsible for the lower skin cancer incidence. We also did not observe any association between other reported risk factors, such as age, human leukocyte antigen mismatch, duration of pretransplant hemodialysis, particular immunosuppressive therapies and the skin cancer occurrence among our kidney recipients.

Possible defect of T suppressor cell subpopulation in patients with kidney acute rejection.

CD8(+)CD28(-) forkhead box P3 (Foxp3(+)) T suppressor (Ts) lymphocytes are antigen-specific cells capable of inducing tolerogenic antigen-presenting cells by up-regulation of inhibitory receptors immunoglobulin-like-transcripts -3 and -4 and down-regulation of costimulatory molecules. Our study sought to investigate the relation between the level of peripheral CD8(+)CD28(-)Foxp3(+) Ts cells and kidney allograft outcomes. The project included 44 kidney transplantation patients. During the 6-month period following transplantation an acute rejection episode (ARE) was diagnosed in 11 patients based on biopsy results using the Banff criteria. Peripheral blood samples collected at 1 day before as well as 14 and 30 days after transplantation were tested for CD8(+)CD28(-)Foxp3(+) T cells by means of flow cytometry. Values were considered significant when P < .05. Cytometric analysis did not show significant differences between the groups in pretransplant levels of peripheral CD8(+)CD28(-)Foxp3(+) Ts cells (P > .05); however, the posttransplantation analysis showed a higher mean level of Ts cells in nonrejection (NONARE) versus acute rejection (ARE) patients (P < .0001). This observation suggested that dysfunction of CD8(+)CD28(-)Foxp3(+) Ts cells observed in ARE patients may contribute to these episodes. Interestingly, we observed similar results with respect to peripheral CD4(+)CD25(+)Foxp3(+) T regulatory cells in ARE patients, suggesting impairment of immunoregulatory mechanisms (especially within the inducible Foxp3 system) in this group, leading to acute renal allograft rejection episodes.

Distinct cytokine patterns in different states of kidney allograft function.

Cytokines are crucial inflammatory mediators involved in the development of immune response leading to allograft rejection. We investigated the cytokine patterns in patients sera from cases of acute rejection episodes (ARE), chronic rejection (CR), and long-term stable courses (STABLE). The project included 20 patients with ARE, 20 with CR, and 15 with at least a 5-year stable course. Serum samples collected at the time of rejection diagnosis were cytometrically tested for concentrations of interleukin (IL) 2, IL-4, IL-6, IL-10, interferon (IFN) gamma, and tumor necrosis factor alpha. No significant differences between investigated groups were observed before transplantation (P > .05). Significant differences were observed among the groups in serum levels of IFN-gamma, IL-4, IL-6, and IL-10. Our data suggested that distinct serum cytokine patterns were present among various states of kidney allograft function. ARE was characterized by a mixed cytokine pattern with elevated IL-10 and IFN-gamma compared with the STABLE patients. The cytokine pattern in CR patients, in turn, was characterized by elevated levels of IL-4, IL-6, and IL-10 and decreased levels of IFN- gamma compared with both STABLE and ARE subjects. Our results suggested that the T(H)2 response may contribute to the initiation and/or maintenance of CR, because IL-4, IL-6, and IL-10 serve as growth and differentiation factors for B cells to increase antibody production. We also observed up-regulated production of IFN-gamma and down-regulation of T(H)2 cytokines among patients with stable long- term graft function.

The role of Foxp3+ regulatory T cells in kidney transplantation.

Our project aimed to investigate the relation between the level of pretransplantation and posttransplantation peripheral CD4(+)CD25(+)Foxp3(+) T-regulatory lymphocytes (Tregs) and the development of acute rejection (AR) episodes in 44 patients after kidney transplantation. During the 6-month period following transplantation, AR was diagnosed in 11 patients. Peripheral blood samples were collected 1 day before and 10 days after transplantation and tested for concentrations of CD4(+)CD25(+)Foxp3(+) cells by means of flow cytometry. The pretransplantation analysis showed significantly lower mean levels of peripheral Tregs in AR patients versus control group (P < .05). A lower level of Tregs was also observed in nonrejection (NONAR) patients versus control group (P < .05); however, it was still higher than in the AR group (P < .05). The 10-day posttransplantation analysis showed a similar pattern; however, a significant increase in the concentration of peripheral Tregs in NONAR patients was observed (P < .05), whereas no change was recorded in AR patients (P > .05). We found lower pretransplantation levels of peripheral Tregs in both AR and NONAR groups, versus control group. The deficiency of peripheral Tregs in patients with end-stage renal failure might be due to the long-term inflammatory processes adversely affecting the peripheral regulatory mechanisms. However, significantly lower levels of Tregs observed in AR patients might also be related to genetic predispositions. Our observation suggests that the size and possibly the functionality of Tregs in the AR group was not sufficient to successfully control the immune response after kidney transplantation, leading to acute rejection episodes.

Role of TH1/TH2 cytokines in kidney allograft rejection.

One of the major issues in contemporary kidney transplantation is prevention of acute allograft rejection episodes (AREs). Cytokines are crucial mediators of immune reactions leading to AREs. We correlated serum Th1/Th2 cytokine concentrations with AREs. The project included 44 patients undergoing kidney transplantation. During the 3-month period following the transplantation, ARE was diagnosed in 11 patients. Serum samples collected 1 day before and 2, 7, 14, and 30 days after transplantation were tested for interleukin (IL)-2, IL-4, IL-5, IL-10, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha concentrations using flow cytometry. Nonrejection (NONAR) and rejection (ARE) groups of patients did not show significant differences in baseline demographic characteristics. We observed that higher pretransplantation serum levels of IFN-gamma (P = .000003) and IL-10 (P = .000001) were associated with AREs. Our analysis also showed slightly higher IL-4 serum levels among NONAR patients up to 7 days posttransplantation, followed by a drop in concentrations in NONAR patients. In contrast, there was a continuous increase among ARE patients. No significant differences were observed in plasma levels of IL-2, IL-5, IL-10, or TNF-alpha between the two groups. Higher pretransplantation levels of IFN-gamma and IL-10 observed in ARE patients indicated ongoing nondetected, probably nonspecific, inflammatory processes able to intensify an immune response directed against the transplanted organ leading to its acute rejection. Higher levels of IL-4 prior to and shortly after transplantation may have protective effects on graft survival. However, a prolonged, increased production of IL-4 after transplantation can also contribute to AREs.

Distribution of the DAZ gene transcripts in human testis.

Involvement of variety of genes, especially located on Y chromosome, is critical for the regulation of spermatogenesis. In particular, fertility candidate genes such as deleted in azoospermia (DAZ) are believed to have important function in sperm production, since DAZ is frequently deleted in azoospermic and severy oligozoospermic men. The role of the DAZ gene is supported by its exclusive expression in the testis and by its deletion in about 10% of azoospermic and severely oligozoospermic patients. The distribution of DAZ transcripts in seminiferous epithelium of human testis is reported in the present study. The use of Adobe Photoshop and Scion Image softwares allowed for semi-quantitative analysis of in situ RT-PCR (ISRT-PCR) results. The intensity of ISRT-PCR product's fluorescence was different within individual seminiferous tubules. It was clearly shown by using the pseudocolour scale and transforming the intensity of the fluorescence into levels of greyscale images. The more intense fluorescence characterised single spermatogonia and those organized in small groups inside separate tubules. The most intense accumulation of DAZ mRNA was observed in spermatogonia.

Vocational rehabilitation following kidney transplantation.

Successful kidney transplantation (KT) improves significantly the quality of life as compared with hemodialysis. Vocational rehabilitation and professional activity are the vital part of general rehabilitation and play an important role in determining the quality of life. The aim of this study was to evaluate the degree of vocational rehabilitation of the patients following allogenic kidney transplantation. 114 patients were questionnaired. Evaluation of the data revealed that only 31.6% of the study group returned to work despite good quality of life and satisfactory graft function of more than 75% of the KT patients. Factors influencing re-employment included gender, age and education level. Most of the patients were re-employed within 6 months following KT. In 78.9% of the cases formal disability status was not changed after KT and remained discrepant with good general condition and graft function. Results of the study allow authors to conclude that unless vocational rehabilitation of the KT patients is not improved, the chances of return to full social life given by KT are mostly wasted.

[Severe hemolytic-uremic syndrome complicated by cholelithiasis treated with laparoscopic cholecystectomy in a 12-year old boy]. / Zespól hemolityczno-mocznicowy o ciezkim przebiegu powiklany kamica pecherzyka zólciowego leczona laparoskopowa cholecystektomia u 12-letniego chlopca.

A case of a 12-year old boy with hemolytic-uremic syndrome complicated with cholelithiasis is presented. Due to severe course of the hemolytic-uremic syndrome (enhanced catabolism, coagulation disorders) a laparoscopic cholecystectomy as a new, modern surgical technique has been considered. Postoperative period has been uncomplicated. Therefore, laparoscopic cholecystectomy proved useful and is recommended in patients with high postoperative risk.

Cytoskeleton association and virion incorporation of the human immunodeficiency virus type 1 Vif protein.

The human immunodeficiency virus type 1 (HIV-1) Vif protein has an important role in the regulation of virus infectivity. This function of Vif is cell type specific, and virions produced in the absence of Vif in restrictive cells have greatly reduced infectivity. We show here that the intracellular localization of Vif is dependent on the presence of the intermediate filament vimentin. Fractionation of acutely infected T cells or transiently transfected HeLa cells demonstrates the existence of a soluble and a cytoskeletal form and to a lesser extent the presence of a detergent-extractable form of Vif. Confocal microscopy suggests that in HeLa cells, Vif is predominantly present in the cytoplasm and closely colocalizes with the intermediate filament vimentin. Treatment of cells with drugs affecting the structure of vimentin filaments affect the localization of Vif accordingly, indicating a close association of Vif with this cytoskeletal component. The association of Vif with vimentin can cause the collapse of the intermediate filament network into a perinuclear aggregate. In contrast, analysis of Vif in vimentin-negative cells reveals significant staining of the nucleus and the nuclear membrane in addition to diffuse cytoplasmic staining. In addition to the association of Vif with intermediate filaments, analyses of virion preparations demonstrate that Vif is incorporated into virus particles. In sucrose density gradients, Vif cosediments with capsid proteins even after detergent treatment of virus preparations, suggesting that Vif is associated with the inner core of HIV particles. We propose a model in which Vif has a crucial function as a virion component either by regulating virus maturation or following virus entry into a host cell possibly involving an interaction with the cellular cytoskeletal network.

Kinetics of in vitro immune responses of T and B cells during tolerance induction by sirolimus.

OBJECTIVES: The purpose of the study presented herein was to examine immune performances of rat heart allograft recipients immunosuppressed with sirolimus (SRL, rapamycin; Rapamune, Wyeth-Ayerst, Princeton, NJ). METHODS: The immune performances of lymphocytes harvested from SRL-treated Wistar Furth (WF; RT1u) recipients of Buffalo (BUF; RT1b) heart allografts were examined on days 7, 14, and 90 postgrafting. RESULTS: Whether derived from normal WF rats, SRL-treated WF heart recipients, or SRL-untreated WF heart recipients, pan-T cell population purified from the lymph nodes or spleens on day 7 or 14 displayed similar responses to phytohemaglutinin, anti-T cell receptor R73 monoclonal antibody, donor-type BUF, or third-party Brown Norway alloantigenic stimulators. There was no in vitro evidence of suppressor T cells in SRL-treated recipients. The frequencies of anti-BUF-specific cytotoxic T cells, as shown by limiting dilution analysis, were similar in the short- (days 7 or 14) and in the long- (day 90) term surviving recipients. SRL treatment did not affect the expression of interleukin-2 (IL-2) messenger RNA (mRNA) by T helper 1 (Th1) or of IL-4 and IL-10 mRNA by Th2 cells on days 7 and 14 postgrafting, but did induce selective activation of Th2 cells on day 60 postgrafting. Administration of SRL induced the production of non-complement (C')-fixing IgG2c BUF-specific alloantibodies that appeared in the sera of unresponsive recipients on day 14 postgrafting and reached a peak concentration on day 120 postgrafting. In contrast to untreated recipients that rejected BUF heart allografts, all SRL-treated WF recipients failed to produce C'-fixing BUF-specific alloantibodies. CONCLUSIONS: SRL promotes long-term selective activation of Th2 cells and the production of non-C'-fixing IgG2c blocking antibodies.

Human immunodeficiency virus type 1 Vpu protein induces degradation of CD4 in vitro: the cytoplasmic domain of CD4 contributes to Vpu sensitivity.

CD4 is an integral membrane glycoprotein which functions as the human immunodeficiency virus (HIV) receptor for infection of human host cells. We have recently demonstrated that Vpu, an HIV type 1 (HIV-1) encoded integral membrane phosphoprotein, induces rapid degradation of CD4 in the endoplasmic reticulum. In this report, we describe an in vitro model system that allowed us to define important parameters for Vpu-dependent CD4 degradation. The rate of CD4 decay in rabbit reticulocyte lysate was approximately one-third of that observed previously in tissue culture experiments in the presence of Vpu (40 versus 12 min) and required no other HIV-1 encoded proteins. Degradation was contingent on the presence of microsomal membranes in the assay and the coexpression of Vpu and CD4 in the same membrane compartment. By using the in vitro degradation assay, the effects of specific mutations in CD4, including C-terminal truncations and glycosylation mutants, were analyzed. The results of these experiments indicate that Vpu has the capacity to induce degradation of glycosylated as well as nonglycosylated membrane-associated CD4. Truncation of 13 C-terminal amino acids of CD4 did not affect the ability of Vpu to induce its degradation. However, the removal of 32 amino acids from the C-terminus of CD4 completely abolished sensitivity to Vpu. This suggests that Vpu targets specific sequences in the cytoplasmic domain of CD4 to induce its degradation. We also analyzed the effects of mutations in Vpu on its biological activity in the in vitro CD4 degradation assay. The results of these experiments suggest that sequences critical for this function of Vpu are located in its hydrophilic C-terminal domain.