Rectus sheath patch. A novel surgical technique in the repair of isolated renal pelvis necrosis in a transplanted kidney. A case report.

INTRODUCTION: Focal necrosis of the renal pelvis in a transplanted kidney is a rare but often morbid complication that may lead to graft loss. Given the scarcity of donor organs, all attempts are made to preserve the graft. Currently there is no standard surgical technique for reconstruction or repair of isolated renal pelvic necrosis. PRESENTATION OF CASE: A 70-year-old male with end stage kidney disease underwent renal transplantation. The patient developed a day-three post-operative urine leak. During surgical exploration, a focal area of pelvic necrosis was observed without evidence of proximal or distal ureteric involvement. Given the excellent function of the renal allograft, a novel surgical technique was successfully used to repair the necrotic defect. Reconstruction of the renal pelvis was performed using an avascular rectus sheath patch. The patch was secured over the open pelvis following necrotic tissue debridement. The patient made a successful recovery with complete resolution of urine leak. A 6-week post-operative retrograde pyelogram confirmed no ongoing urine leak. DISCUSSION: To restore anatomy, the pelvic defect was patched with avascular rectus sheath fascia. Advantages of this reconstructive method were technique simplicity and low donor site morbidity. Potential complications included patch failure with ongoing urine leak, ventral wall hernia through the fascial donor site and stenosis of the ureteropelvic junction. CONCLUSION: This case highlights the successful surgical management of a renal pelvis urine leak patched with rectus sheath fascia. This technique could be considered as a graft saving procedure in similar cases where the alternative is transplant nephrectomy.

Urgent nephrectomy for rapidly progressive disseminated metastatic Hypermucoid Klebsiella.

Few cases of Hypervirulent Klebsiella Pneumonia (HvKP) have been described. Even fewer cases with renal abscess and metastatic pulmonary spread are reported. Typically, prompt introduction of intravenous antibiotics leads to clinical resolution and more invasive measures of source control are rarely required. To date only one other case of disseminated metastatic HvKP requiring nephrectomy for infective source control is described. Here we present a rare case of metastatic HvKP refractory to intravenous antimicrobial therapy in an immunocompromised newly diagnosed diabetic patient. Specifically, we seek to illustrate the rapid effectiveness of surgical intervention following a poor response to initial treatment.

Cryptosporidium homai n. sp. (Apicomplexa: Cryptosporidiiae) from the guinea pig (Cavia porcellus).

The morphological, biological, and molecular characterisation of a new Cryptosporidium species from the guinea pig (Cavia porcellus) are described, and the species name Cryptosporidium homai n. sp. is proposed. Histological analysis conducted on a post-mortem sample from a guinea pig euthanised due to respiratory distress, identified developmental stages of C. homai n. sp. (trophozoites and meronts) along the intestinal epithelium. Molecular analysis at 18S rRNA (18S), actin and hsp70 loci was then conducted on faeces from an additional 7 guinea pigs positive for C. homai n. sp. At the 18S, actin and hsp70 loci, C. homai n. sp. exhibited genetic distances ranging from 3.1% to 14.3%, 14.4% to 24.5%, and 6.6% to 20.9% from other Cryptosporidium spp., respectively. At the 18S locus, C. homai n. sp. shared 99.1% similarity with a previously described Cryptosporidium genotype in guinea pigs from Brazil and it is likely that they are the same species, however this cannot be confirmed as actin and hsp70 sequences from the Brazilian guinea pig genotype are not available. Phylogenetic analysis of concatenated 18S, actin and hsp70 sequences showed that C. homai n. sp. exhibited 9.1% to 17.3% genetic distance from all other Cryptosporidium spp. This clearly supports the validity of C. homai n. sp. as a separate species.

Pilot study: use of gallium-68 PSMA PET for detection of metastatic lesions in patients with renal tumour.

BACKGROUND: In this study, we prospectively evaluate the diagnostic potential of a gallium-68 (68Ga) prostate-specific membrane antigen (PSMA)-binding ligand and positron emission tomography (PET) in detecting metastatic lesions in patients with renal tumour. The secondary aim was to determine whether the findings would result in the alteration of patient management. RESULTS: Ten patients with renal lesion and potential metastatic disease on conventional imaging were recruited. Patients underwent PSMA PET in addition to standard imaging. Nine patients underwent nephrectomy and 4 patients underwent additional targeted biopsy to provide specimens for histopathological validation. There were 89 pathological lesions on CT, of which 32 were removed or biopsied for histopathological correlation. With PSMA PET, 86 PET avid lesions were identified with 36 samples being available for analysis. Thirty-five of 36 samples were positive for renal cell carcinoma deposits, whilst 1 sample was inconclusive for diagnosis on biopsy. For the histologically confirmed lesions, there were no false-negative PSMA PET lesions; however, CT was false negative in 11. In two patients, surgical strategies were changed based on PSMA PET findings. CONCLUSIONS: PSMA PET may potentially have a role in the preoperative staging of advanced renal cell carcinoma as PET detected multiple histologically proven metastatic lesions which were false negative on CT scanning, resulting in change in surgical strategies in some patients. We cautiously support a larger study to confirm these results and to assess the longitudinal impact on patient outcomes. TRIAL REGISTRATION: Australia and New Zealand Clinical Trial Registry (ANZCTR), ACTRN12615000854538 .

Key contribution of eIF4H-mediated translational control in tumor promotion.

Dysregulated expression of translation initiation factors has been associated with carcinogenesis, but underlying mechanisms remains to be fully understood. Here we show that eIF4H (eukaryotic translation initiation factor 4H), an activator of the RNA helicase eIF4A, is overexpressed in lung carcinomas and predictive of response to chemotherapy. In lung cancer cells, depletion of eIF4H enhances sensitization to chemotherapy, decreases cell migration and inhibits tumor growth in vivo, in association with reduced translation of mRNA encoding cell-proliferation (c-Myc, cyclin D1) angiogenic (FGF-2) and anti-apoptotic factors (CIAP-1, BCL-xL). Conversely, each isoform of eIF4H acts as an oncogene in NIH3T3 cells by stimulating transformation, invasion, tumor growth and resistance to drug-induced apoptosis together with increased translation of IRES-containing or structured 5'UTR mRNAs. These results demonstrate that eIF4H plays a crucial role in translational control and can promote cellular transformation by preferentially regulating the translation of potent growth and survival factor mRNAs, indicating that eIF4H is a promising new molecular target for cancer therapy.

Two novel determinants of etoposide resistance in small cell lung cancer.

Patient survival in small cell lung cancer (SCLC) is limited by acquired chemoresistance. Here we report the use of a biologically relevant model to identify novel candidate genes mediating in vivo acquired resistance to etoposide. Candidate genes derived from a cDNA microarray analysis were cloned and transiently overexpressed to evaluate their potential functional roles. We identified two promising genes in the DNA repair enzyme DNA polymerase ß and in the neuroendocrine transcription factor NKX2.2. Specific inhibition of DNA polymerase ß reduced the numbers of cells surviving treatment with etoposide and increased the amount of DNA damage in cells. Conversely, stable overexpression of NKX2.2 increased cell survival in response to etoposide in SCLC cell lines. Consistent with these findings, we found that an absence of nuclear staining for NKX2.2 in SCLC primary tumors was an independent predictor of improved outcomes in chemotherapy-treated patients. Taken together, our findings justify future prospective studies to confirm the roles of these molecules in mediating chemotherapy resistance in SCLC.

Tissue banking of diagnostic lung cancer biopsies for extraction of high quality RNA.

INTRODUCTION: There is a clear need to develop a practical approach to obtain high quality RNA for gene expression analysis from lung cancer patients. Current approaches are restricted to using material from surgical resection specimens. We systematically investigated whether high quality RNA could be obtained from routine lung cancer diagnostic biopsies to determine the optimum method. METHODS: Extra biopsies were taken at diagnosis from patients later confirmed to have lung cancer. Comparisons were made between RNA extracted from samples snap frozen in liquid nitrogen and those treated with an RNA preservative before freezing. Further comparisons were made between biopsies taken by different methods. RESULTS: Acceptable RNA for gene expression analysis was extracted from 72% of lung cancer biopsies. Use of an RNA preservative for storage allowed the extraction of higher quality, more intact RNA from biopsies gathered by both endobronchial forceps and transbronchial needle aspiration. High quality RNA could also be extracted from computed tomography-guided needle core biopsies. CONCLUSION: Banking lung cancer biopsy specimens by storage in an RNA preservative solution will allow use of a broader spectrum of lung cancers for gene expression analysis. We describe a model that makes personalized medicine for lung cancer patients a more practical proposition.

Promoter control over foreign antigen expression in a murine cytomegalovirus vaccine vector.

Previous studies have reported on the development of a recombinant murine cytomegalovirus (rMCMV) containing the mouse zona pellucida 3 (mZP3) gene for use as a virally vectored immunocontraceptive (VVIC). This study aimed to alter promoter control over foreign antigen expression and cellular localisation of the antigen expressed in order to overcome virus attenuation previously encountered. Early studies reported on the mZP3 gene expressed by a strong constitutive human cytomegalovirus immediate-early 1 promoter (pHCMV IE1). This virus was able to induce >90% infertility in BALB/c mice despite being heavily attenuated in vivo. In this study the mZP3 was placed under the control of the MCMV early 1 (pMCMV E1) promoter and the inducible tetracycline promoter (Tet-On). In both instances the recombinant virus was able to induce infertility in directly infected mice. However, the viruses remained attenuated. This study demonstrated the capacity to manipulate the nature of the immune response by altering promoter control over foreign antigen expression and cellular localisation of the expressed antigen. We were able to demonstrate that by using the MCMV E1 promoter it was still possible to sterilize female BALB/c mice with an MCMV vector expressing mZP3. The use of the MCMV E1 promoter provides an added level of safety to any MCMV based VVIC approach as it only allows for transgene expression in MCMV permissive cells.

Urological implications of cyclophosphamide and ifosfamide.

Cyclophosphamide and ifosfamide are commonly used cytotoxic medications that are indicated in a wide range of conditions, both benign and malignant. Complications of their use include well-recognized acute and chronic urological side-effects. Haemorrhagic cystitis, nephrotoxicity and transitional cell carcinoma can be directly attributed to the use of these agents, and are potentially fatal. Preventive measures can be used in an attempt to minimize the rate of complications. Urological intervention may be required in the acute and long-term management of patients who have received oxazaphosphorine agents. This article reviews current literature, sourced using a MEDLINE search of the keywords with cross-referencing. All articles were reviewed by abstract. Selection of articles was on the basis of randomized controlled trials and articles adding new or significant information.

Prior infection with murine cytomegalovirus (MCMV) limits the immunocontraceptive effects of an MCMV vector expressing the mouse zona-pellucida-3 protein.

We have developed a murine cytomegalovirus (MCMV)-vectored vaccine expressing the mouse zona-pellucida-3 gene (rMCMV-ZP3), which successfully induces infertility in experimentally inoculated laboratory or wild-derived mice. However, the future success of this vector as a fully disseminating vaccine in free-living mice may be compromised by pre-existing immunity since there is a high prevalence of naturally acquired MCMV infection in these mice. To evaluate the effect of prior immunity to MCMV on vaccine efficacy, we constructed two new biologically effective recombinant MCMV vectors expressing the mouse ZP3 protein from two MCMV strains (N1 and G4) derived from free-living mice. In wild mice, mixed MCMV infection is common and could be acquired either by simultaneous coinfection or sequential infection with different MCMV strains. Interestingly, while coinfection with both wild-type and rMCMV-ZP3 via the intraperitoneal route reduced the impact of the rMCMV-ZP3, prior infection with the same wild-type strain as that used to construct the rMCMV-ZP3 abrogated the immunocontraceptive effects of either N1-ZP3 or G4-ZP3. However, prior infection with G4 28 days before the introduction of N1-ZP3 had a reduced influence on the efficacy of the rMCMV-ZP3. Thus, the strain of virus and the timing of prior infection are factors that may influence the efficacy of the rMCMV-ZP3. Given that mixed infection of mice with MCMV is common, it is possible that prior immunity acquired by natural mucosal infection may have less a less inhibitory effect on the immunocontraceptive outcome.

Innate antiviral resistance influences the efficacy of a recombinant murine cytomegalovirus immunocontraceptive vaccine.

Recombinant betaherpesviruses are attractive vaccine candidates because of their persistence in the host. A recombinant murine cytomegalovirus expressing the mouse ovarian glycoprotein zona pellucida 3 induces long lasting sterility in female BALB/c mice. Using inbred mouse strains selected for their innate resistance or susceptibility to MCMV, we show that genetically determined innate resistance to MCMV can reduce immunocontraceptive success. The Cmv1 locus that controls natural killer cell mediated responses to MCMV was implicated in determining vaccine efficacy. However, the role of the H-2 haplotype was less clear. Interestingly, Mus domesticus from an outbred colony of wild-derived mice were readily sterilised by vaccination, consistent with observations that strong innate immunity to MCMV is not common in Australian wild mice.

Viral vectored immunocontraception: screening of multiple fertility antigens using murine cytomegalovirus as a vaccine vector.

Mouse cytomegalovirus (MCMV) has previously been used as a vaccine vector for viral vectored immunocontraception (VVIC). MCMV expressing murine zona pellucida 3 (mZP3) induces long term infertility in up to 100% of female BALB/c mice following a single inoculation. Whilst a large number of antigens have been investigated as potential immunocontraceptive vaccines, it has been difficult to compare these antigens as few studies have used identical approaches or even animal species. Here a range of protein and polyepitope antigens, all expressed by MCMV, were tested for the ability to sterilise female mice. The antigens tested were bone morphogenic protein 15 (BMP15), oviduct glycoprotein (OGP) and ubiquitin-tagged mZP3. In addition, four polyepitope constructs that contain rodent or mouse specific epitopes were tested. This study found that when expressed by an MCMV vector, only full-length mZP3 or ubiquitin-tagged mZP3 induced infertility in female mice. BMP15 and OGP had no effect. Of the four polyepitopes tested, one had a partial effect on fertility. These data indicate that while MCMV is an effective vector for VVIC, the antigen used needs to be tested empirically. The partial infertility seen in mice infected with one of the polyepitope vaccines is a promising finding suggesting that it may be possible to combine a species specific virus with a species specific antigen for use as a disseminating mouse control agent.

Mixed infection with multiple strains of murine cytomegalovirus occurs following simultaneous or sequential infection of immunocompetent mice.

As with human cytomegalovirus (HCMV) infection of humans, murine CMV (MCMV) infection is widespread in its natural host, the house mouse Mus domesticus, and may consist of mixed infection with different CMV isolates. The incidence and mechanisms by which mixed infection occurs in free-living mice are unknown. This study used two approaches to determine whether mixed infection with MCMV could be established in laboratory mice. The first utilized two naturally occurring MCMV strains, N1 and G4, into which the lacZ gene was inserted by homologous recombination. The lacZ gene was used to track recombinant and parental viruses in simultaneously coinfected mice. In the second approach, a real-time quantitative PCR (qPCR) assay was used to detect viral immediate-early 1 (ie1) gene sequences in mice successively coinfected with G4 and then with the K181 MCMV strain. In both systems, mixed infection was detected in the salivary glands and lungs of experimentally infected mice. MCMV-specific antibody in sera and G4 IE1-specific cytotoxic lymphocyte responses in the spleens of twice-infected mice did not prevent reinfection. Finally, the prevalence of mixed infection in free-living mice trapped in four Australian locations was investigated using real-time qPCR to detect ie1 DNA sequences of N1, G4 and K181. Mixed infection with MCMVs containing the G4 and K181 ie1 sequences was detected in the salivary glands of 34.2 % of trapped mice. The observations that mixed infections are common in free-living M. domesticus and are acquired by immunocompetent mice through simultaneous or successive infections are important for vaccine development.

Use of a murine cytomegalovirus K181-derived bacterial artificial chromosome as a vaccine vector for immunocontraception.

Cytomegaloviruses (CMVs) are members of the Betaherpesvirinae subfamily of the Herpesviridae, and their properties of latency, large DNA size, gene redundancy, and ability to be cloned as bacterial artificial chromosomes (BACs) suggest their utility as vaccine vectors. While the K181 strain of murine CMV (MCMV) is widely used to study MCMV biology, a BAC clone of this virus had not previously been produced. We report here the construction of a BAC clone of the K181(Perth) strain of MCMV. The in vivo and in vitro growth characteristics of virus derived from the K181 BAC were similar to those of wild-type K181. The utility of the K181 BAC as a method for the rapid production of vaccine vectors was assessed. A vaccine strain of BAC virus, expressing the self-fertility antigen, murine zona pellucida 3, was produced rapidly using standard bacterial genetics techniques and rendered female BALB/c mice infertile with a single intraperitoneal inoculation. In addition, attenuated vaccine strains lacking the open reading frames m07 to m12 exhibited no reduction in efficacy compared to the full-length vaccine strain. In conclusion, we describe the production of a K181-based BAC virus which behaved essentially as wild-type K181 and allowed the rapid production of effective viral vaccine vectors.

Assessment of contraceptive vaccines based on recombinant mouse sperm protein PH20.

Mouse PH20 (mPH20), the mouse homologue to guinea pig hyaluronidase protein PH20 (gpPH20), was used to produce contraceptive vaccines that target both sexes of mice. Previously, immunization with a female gamete antigen (the zona pellucida subunit 3 protein) delivered in a recombinant murine cytomegalovirus (MCMV), or as a purified recombinant protein, has been shown to induce infertility in female mice. There is evidence, however, that sperm protein antigens could provide broader contraceptive coverage by affecting both males and females, and the most promising has been gpPH20 when tested in a guinea pig model. Mice were therefore either inoculated with a recombinant MCMV expressing mPH20 or immunized directly with purified recombinant mPH20 protein fused to maltose-binding protein. Mice treated with either vaccine formulation developed serum antibodies that cross-reacted to a protein band of 55 kDa corresponding to mPH20 in Western blots of mouse sperm. However, there was no significant reduction in the fertility of males or females compared with control animals with either formulation. We conclude from our data that recombinant mPH20 is not a useful antigen for inclusion in immunocontraceptive vaccines that target mice.

Immunocontraception is induced in BALB/c mice inoculated with murine cytomegalovirus expressing mouse zona pellucida 3.

Immunocontraception, the prevention of oocyte fertilization through immunological means, could potentially be used to control plaguing mouse populations in Australia. This paper describes the construction of a mouse-specific betaherpesvirus, murine cytomegalovirus, which has been engineered to express the murine zona pellucida 3 (ZP3) gene. A single inoculation of this recombinant virus resulted in almost complete infertility, persistent anti-ZP3 antibody production, and profound changes to ovarian morphology in BALB/c mice in the absence of significant virus replication during the acute phase of infection. Murine cytomegalovirus may prove to be useful as a vector for the delivery of a mouse-specific immunocontraceptive agent to target populations of wild mice in the field.