Low-dose carboplatin modifies the tumor microenvironment to augment CAR T cell efficacy in human prostate cancer models.

Chimeric antigen receptor (CAR) T cells have transformed the treatment landscape for hematological malignancies. However, CAR T cells are less efficient against solid tumors, largely due to poor infiltration resulting from the immunosuppressive nature of the tumor microenvironment (TME). Here, we assessed the efficacy of Lewis Y antigen (LeY)-specific CAR T cells in patient-derived xenograft (PDX) models of prostate cancer. In vitro, LeY CAR T cells directly killed organoids derived from androgen receptor (AR)-positive or AR-null PDXs. In vivo, although LeY CAR T cells alone did not reduce tumor growth, a single prior dose of carboplatin reduced tumor burden. Carboplatin had a pro-inflammatory effect on the TME that facilitated early and durable CAR T cell infiltration, including an altered cancer-associated fibroblast phenotype, enhanced extracellular matrix degradation and re-oriented M1 macrophage differentiation. In a PDX less sensitive to carboplatin, CAR T cell infiltration was dampened; however, a reduction in tumor burden was still observed with increased T cell activation. These findings indicate that carboplatin improves the efficacy of CAR T cell treatment, with the extent of the response dependent on changes induced within the TME.

Women's experience of unexpected caesarean section birth in Kitui County, Kenya.

Background: In sub-Saharan Africa, 72% of all Caesarean section (CS) births are unplanned compared to 27% of unplanned CS births done in developed countries. Various researches have been conducted on lived experiences following unexpected CS birth but none in Kitui County, Kenya. Objective: This study described the lived experience of undergoing an unexpected CS and the role of cultural beliefs on childbirth among Kitui/Kamba women in Kenya. Methods: A descriptive phenomenology design was used in this study. In-depth interviews of women who experienced unplanned Caesarean birth in Kitui County, Kenya, were conducted. Colaizzi's method guided the analysis. Interviews were approximately 30 minutes long and audiotaped. Results: A total of 12 mothers participated in the study. Fives themes and 11 sub-themes emerged from this study: fear (fear of disability and surgical complications), pain (physical and psychological pain), less of a woman (lowered self-esteem, powerless and worry), sullied (dispirited and will loss), and fallacy (misconception and effects of fallacy). Conclusion: Disruption, dissatisfaction with the birth process, and unmet expectations were negative experiences. Healthcare workers should be sensitive when informing mothers of unplanned CS. More research to look for coping strategies to reduce negative birthing experiences.

Dietary quality and carotid intima media thickness in type 1 and type 2 diabetes: Follow-up of a randomised controlled trial.

BACKGROUND AND AIMS: The relationship between dietary intake and carotid intima media thickness (IMT) and pulse wave velocity (PWV) in individuals with type 1 and type 2 diabetes has not been well studied. We investigated the association between dietary intake and common carotid artery intima media thickness (CCA IMT) and PWV in a cohort with type 1 and type 2 diabetes. METHODS AND RESULTS: A one-year randomised controlled trial was conducted to investigate the effect of improving dietary quality on CCA IMT. These subjects were followed up again approximately 12 months after the completion of the trial (i.e. approximately 24 month since baseline). The study cohort included 87 subjects that had dietary intake and CCA IMT measured at baseline and after a mean of 2.3 years' follow-up. PWV was measured in a subsample of this cohort. Age and baseline mean CCA IMT were strongly associated with mean CCA IMT at 24 months. After adjustment for age and baseline mean CCA IMT, baseline consumption of carbohydrate (r = -0.28; p = 0.01), sugars (r = -0.27; p = 0.01), fibre (r = -0.26; p = 0.02), magnesium (r = -0.25; p = 0.02) and the Alternate Health Eating Index (AHEI) score (r = -0.23; p = 0.03) were inversely associated with mean CCA IMT at 24 months. Mixed linear modelling showed an interaction between mean CCA IMT and AHEI at baseline (p = 0.024). Those who were in the highest AHEI tertile at baseline had greater CCA IMT regression at 24 months compared to those in the lowest tertile, after adjustment for baseline age, BMI, smoking pack years, time since diabetes diagnosis, and mean arterial pressure at baseline (mean -0.043 mm; 95% CI -0.084, -0.003; p = 0.029). CONCLUSIONS: In this prospective analysis greater diet quality at baseline, as measured by the AHEI, was associated with greater CCA IMT regression after approximately two years. This suggests that greater diet quality is associated with better longer term vascular health in individuals with type 1 and type 2 diabetes.

Nutritional adequacy of diets for adolescents with overweight and obesity: considerations for dietetic practice.

BACKGROUND/OBJECTIVES: Adolescents have unique nutrient requirements due to rapid growth and development. High rates of obesity in adolescents require a variety of diet interventions to achieve weight loss under clinical supervision. The aim of this study is to examine the nutritional adequacy of energy-restricted diets for adolescents. SUBJECTS/METHODS: Three popular diets were modelled for 7 days and assessed by comparing the nutrient profile to the Australian Nutrient Reference Values. Three diets were: (1) a standard energy restricted diet based on current dietary guidelines; (2) a modified carbohydrate diet; and (3) a modified alternate day fasting diet. RESULTS: Initial modelling revealed limiting nutrients (that is, not meeting the recommended intakes) across the diets. Subsequent modelling was required to achieve nutritional adequacy for all three diets. The dietary guidelines diet design met most nutrient targets except essential fatty acids before subsequent modelling, however this diet also provided the highest energy (8.8 vs 8.0 MJ and 6.8 MJ for the modified carbohydrate and modified alternate day fasting diet, respectively). CONCLUSIONS: Energy-restricted diets need careful consideration to meet nutritional requirements of adolescents. A variety of eating patterns can be adapted to achieve nutritional adequacy and energy restriction, however health practitioners need to consider adequacy when prescribing diet interventions for weight loss during adolescence.

Effect of weight loss induced by energy restriction on measures of arterial compliance: A systematic review and meta-analysis.

AIM: To conduct a systematic review and meta-analysis of clinical trials involving adults, to determine the effect of weight loss induced by energy restriction with or without exercise, anti-obesity drugs or bariatric surgery on measures of arterial stiffness and compliance. METHODS: A systematic search of Pubmed, EMBASE, MEDLINE and the Cochrane Library was conducted to find intervention trials (randomised/non-randomised) that aimed to achieve weight loss and included the following outcome measures: cardio-ankle vascular index (CAVI), direct measures of area/diameter related to pressure change (including ß-stiffness index, brachial or carotid artery compliance, aortic, carotid or brachial artery distensibility and strain), measures derived from peripheral pulse wave analysis (including augmentation index, augmentation pressure, distal oscillatory, proximal capacitive and systemic compliance) and pulse pressure. Data were analysed using Comprehensive Meta Analysis V2 using random effects analysis. Standardised mean difference (SMD) is reported with negative values indicating an improvement. RESULTS: A total of 43 studies, involving 4231 participants, were included in the meta-analysis. Mean weight loss was approximately 11% of initial body weight. Weight loss improved CAVI (SMD -0.48; p = 0.04), ß-stiffness index (SMD = -0.98; p = 0.001), arterial compliance (SMD = -0.61; p = 0.0001) and distensibility (SMD -1.10; p = 0.005), distal oscillatory compliance (SMD = -0.41; p = 0.03), proximal capacitive compliance (SMD -0.66; p = 0.009), systemic arterial compliance (SMD -0.71; p = 0.003) and reflection time (SMD -0.51; p = 0.001). Augmentation index, strain, augmentation pressure and pulse pressure were not significantly changed with weight loss. CONCLUSION: Weight loss induced by energy restriction improves some measures of arterial compliance and stiffness.

Establishment of transplantation tolerance via minimal conditioning in aged recipients.

Mixed hematopoietic chimerism is a powerful means of generating donor-specific tolerance, allowing long-term graft acceptance without lifelong dependence on immunosuppressive drugs. To avoid the need for whole body irradiation and associated side effects, we utilized a radiation-free minimal conditioning regime to induce long-term tolerance across major histocompatibility barriers. We found that low-dose busulfan, in combination with host T cell depletion and short-term sirolimus-based immunosuppression, facilitated efficient donor engraftment. Tolerance was achieved when mice were transplanted with whole or T cell-depleted bone marrow, or purified progenitor cells. Tolerance induction was associated with an expansion in regulatory T cells and was not abrogated in the absence of a thymus, suggesting a dominant or compensatory peripheral mode of tolerance. Importantly, we were able to generate durable chimerism and tolerance to donor skin grafts in both young and aged mice, despite age-related thymic atrophy and immune senescence. Clinically, this is especially relevant as the majority of transplant recipients are older patients whose immune recovery might be dangerously slow and would benefit from radiation-free minimal conditioning regimes that allow efficient donor engraftment without fully ablating the recipient immune system.

Mycoplasma genitalium in men who have sex with men at male-only saunas.

OBJECTIVES: To determine the prevalence and clinical associations of Mycoplasma genitalium in urethral, rectal and pharyngeal specimens collected from men who have sex with men (MSM) attending male-only saunas. METHODS: A cross-sectional study of 521 MSM attending six male-only saunas in Melbourne was conducted between October 2001 and September 2002. Stored urine and rectal and pharyngeal swabs were tested for M genitalium by real-time polymerase chain reaction. The prevalence of M genitalium and clinical associations were determined. RESULTS: A high prevalence (12.9%; 95% CI 10.2% to 15.9%) of sexually transmitted infections (STIs) was found in MSM. M genitalium (2.1%; 95% CI 1.1% to 3.6%) was less common than Chlamydia trachomatis (8.1%; 95% CI 5.9% to 10.6%, p<0.001), and Neisseria gonorrhoeae (4.8%; 95% CI 3.2% to 6.9%, p = 0.02). M genitalium was most likely to be detected as an asymptomatic rectal (1.6%; 95% CI 0.8% to 3.0%) or urethral infection (0.6%; 95% CI 0.2% to 1.6%, p = 0.12), but was absent from the pharynx. In comparison, C trachomatis was more common in the rectum (6.2%; 95% CI 4.3% to 8.6%) than urethra (1.8%; 95% CI 0.9% to 3.2%, p = 0.004), and was uncommon in the pharynx (0.6%; 95% CI 0.1% to 1.6%). Urethral infection with N gonorrhoeae (0.2%; 95% CI 0.01% to 1.0%) was rare, but it was as common in the pharynx (2.5%; 95% CI 1.4% to 4.2%) as the rectum (2.2%; 95% CI 1.2% to 3.8%). No significant demographic or behavioural associations with M genitalium were identified. CONCLUSION: M genitalium was less common than C trachomatis and N gonorrhoeae in MSM attending male-only saunas and was most often detected as an asymptomatic rectal or a urethral infection but was absent from the pharynx. To inform STI screening strategies in MSM, more data are needed to understand how common M genitalium infection is in urethral and non-urethral sites in MSM, and how it contributes to clinical symptoms.

Evaluation of PorB variable region typing of Neisseria gonorrhoeae using PCR-ELISA in samples collected from men who have sex with men.

A polymerase chain reaction (PCR)-based enzyme-linked immunosorbent assay (ELISA) methodology was developed to characterize Neisseria gonorrhoeae porB gene variable regions (VR); the methodology was evaluated in comparison to porB VR typing by checkerboard hybridization. Clinical noncultured samples from 35 men who have sex with men (MSM), positive by nucleic amplification assays for N. gonorrhoeae, were typed using a panel of 40 oligonucleotide probes to porB VRs and compared to checkerboard hybridization. Complete concordance was observed between the two methods at PIB VRs 1, 3, and 7. At the more degenerate VRs 5 and 6, PCR ELISA resulted in obtaining more typeable VRs than checkerboard hybridization due to single nucleotide mismatches. By PCR ELISA, two predominant PIB porB types were identified in 58% of the samples and the remaining 16 samples had one of six other porB types. Both PCR ELISA and checkerboard hybridization methods of porB VR typing allowed characterization of N. gonorrhoeae from noncultured clinical samples including throat and rectal swabs and discriminated N. gonorrhoeae from N. meningitidis present in some of the samples. PCR ELISA is a rapid, relatively inexpensive and alternative molecular typing method for N. gonorrhoeae, suitable for use in conjunction with molecular diagnostic tests.

Association between risk of acquiring HIV and beliefs and perceptions about the lived experience of HIV/AIDS among HIV-negative or untested men who have sex with men.

The study aim was to assess whether the sexual behaviour of HIV-negative or untested men who have sex with men (MSM) was related to their perceptions of what it is like to live with HIV/AIDS, their beliefs or their attitudes to highly active antiretroviral treatments. Any unprotected anal intercourse (UAI) with casual partners was used as the sexual-risk indicator. The study enrolled 261 MSM. There were no significant differences between beliefs, attitudes and perceptions about HIV/AIDS, knowledge of post-exposure prophylaxis (PEP) or exposure to the HIV/AIDS epidemic among those who had had UAI with casual partners and those that had not (P>0.12). Those who considered that low levels of viral load and withdrawing before ejaculation reduced the risk of HIV transmission were significantly more likely to have had UAI with a casual partner (P=0.03). Only a minority of MSM engaging in UAI were optimistic about antiretroviral therapy. The study participants were in general pessimistic about life with HIV/AIDS despite their risk-taking sexual behaviour.

Re-infection of Neisseria gonorrhoeae and Chlamydia trachomatis infections among men who have sex with men.

Our aim was to determine the incidence of re-infection of men who have sex with men (MSM) for rectal Neisseria gonorrhoeae (RNg) and rectal Chlamydia trachomatis (RCt) infection. The medical records of 126 MSM diagnosed with RNg or RCt infections at Melbourne Sexual Health Centre were reviewed. A total of 68 of the 126 (54%) MSM were re-tested. Among the HIV-positive MSM, 17 (81%) of 21 were re-tested and eight were positive (47%, 95% confidence interval [CI] 23-72%). Among the 51 (49%) of 105 of the HIV-negative or unknown MSM re-tested, 13 (25%, 14-40) were positive. Of the 21 infections identified, five cases were symptomatic. The incidence of rectal infections among MSM is high and prior RNg or RCt should be included as a risk factor for frequent screening.

Increased acute GvHD and higher transplant-related mortality in non-caucasians undergoing standard sibling allogeneic stem cell transplantation.

We conducted a retrospective study to compare outcome in Caucasians and non-Caucasians undergoing standard sibling allogeneic SCT. End points of the study were to compare graft-versus-host disease (GvHD) occurrence and transplant-related mortality (TRM). There were 251 patients, 43 non-Caucasian and 208 Caucasian. A higher proportion of non-Caucasian patients developed acute GvHD (aGvHD) grade 2 or greater as compared to the Caucasian group (48 vs 26%, respectively) P = 0.02. With a median follow-up of 27 months, 26% (11/43) of non-Caucasians and 14% (29/208) of Caucasian patients had died from TRM, which accounted for 55% of all deaths in the non-Caucasian group compared to 33% in Caucasians, P = 0.02. Overall survival 12 months post transplant was 64 vs 69% in the non-Caucasian and Caucasian groups, respectively (P = 0.43). Although there were higher numbers of CMV-positive patients in the non-Caucasian group, there were no deaths from CMV reactivation in this subgroup. We conclude that there is increased TRM and aGvHD following standard sibling allograft in the non-Caucasian population and this could be due to either differences in tumour biology or extrinsic factors such as socio-economic factors, nutritional status, post transplant care or presenting with late stage disease.

Validation of roche COBAS Amplicor assay for detection of Chlamydia trachomatis in rectal and pharyngeal specimens by an omp1 PCR assay.

Screening guidelines for men who have sex with men (MSM) recommend testing of extragenital sites (pharyngeal and rectal) for gonorrhoea and chlamydia. Testing of specimens from these sites is not validated by most commercial nucleic amplification tests, such as the COBAS Amplicor assay. To investigate the utility of the COBAS Amplicor assay for detection of Chlamydia trachomatis in extragenital specimens, this study developed and evaluated confirmatory tests using the omp1 gene as an alternative target for amplification by PCR. Of anal and throat swabs collected from men in male-only saunas, 52 swabs that tested C. trachomatis positive by COBAS Amplicor and 30 swabs that tested as negative were included for confirmatory omp1 PCR testing. A total of 49 (94%) COBAS Amplicor-positive samples were confirmed by the omp1 PCR. A substantial proportion of specimens were confirmed by using a nested omp1 PCR (27%). Not confirmed by any omp1 PCR were three anal swabs (6%). It is most probable that these samples contained lower bacterial levels that were near or below the detection level of the omp1 PCR assays. The findings of this study support the confident reporting of C. trachomatis detected by COBAS Amplicor in extragenital specimens and support the utility of this assay as a screening test for MSM.

Testing men who have sex with men for Neisseria gonorrhoeae and Chlamydia trachomatis prior to the introduction of guidelines at an STD clinic in Melbourne.

BACKGROUND: Guidelines for testing men who have sex with men (MSM) were published in 2002. They did not recommend asymptomatic screening for urethral gonorrhoea or pharyngeal screening for chlamydia. To determine if these guidelines were appropriate, we audited gonorrhoea and chlamydia testing of MSM at our Centre. METHODS: We carried out two audits at our Centre between August 2001 and July 2002. The first was an audit of testing MSM for gonorrhoea and/or chlamydia over 12 days. The second was an audit of all positive tests over this 12-month period for gonorrhoea or chlamydia among MSM. RESULTS: During the 12 selected days 89 of 286 men tested (31%) were MSM. Among the MSM testing positive for gonorrhoea and/or chlamydia infection (15, 17%), symptomatic urethral infection was the most common (n = 8). No rectal and pharyngeal infections had site-specific symptoms. Based on the guidelines, 100 of the 334 tests ordered (30%) were not recommended according to the guidelines, and none of these 100 tests yielded a positive result. Over the 12-month audit period, 135 MSM were diagnosed with gonorrhoea and/or chlamydia. For gonorrhoea, site specific symptoms were present in 42 of 43 cases of urethral infection (98%), six of 23 cases of rectal infection (26%), and no cases of pharyngeal infection had symptoms. For chlamydia, site-specific symptoms were present in 29 of 48 cases of urethral infection (60%), six of 33 cases of rectal infection (18%), and in one of the two cases of pharyngeal chlamydia identified. A substantial proportion of cases occurred in clients with HIV infection (21, 16%). CONCLUSIONS: These findings strongly support screening among MSM and in particular not testing asymptomatic MSM for urethral gonorrhoea or any MSM for pharyngeal chlamydia.

Screening for Neisseria gonorrhoeae and Chlamydia trachomatis in men who have sex with men at male-only saunas.

BACKGROUND AND OBJECTIVES: In response to increases in sexually transmissible infections (STI) and HIV infection rates among men who have sex with men (MSM), the current study aimed to investigate the feasibility of a screening program at male-only saunas in Melbourne, Australia. GOAL: The goal was to determine (1) the participation rate, and the proportion whom obtain test results; (2) the prevalence of gonorrhea and chlamydia; and (3) to evaluate risk factors for STI acquisition. STUDY DESIGN: We used a cross-sectional design. Pharyngeal, rectal, and urethral specimens were collected from participants, and tested for Chlamydia trachomatis and Neisseria gonorrhoeae by polymerase chain reaction (PCR). RESULTS: There was a participation rate of 24% (n=521), and 70% obtained their test results. The infection rate in those who failed to collect their results was no different than those seeking theirs. The proportion of participants with PCR-detected gonorrhea and/or chlamydia infection was high, 10.7%. The presence of infection was associated with seeking sexual health care in the last year. CONCLUSION: The high prevalence rate of gonorrhea and chlamydia supports the concept of a screening program in Melbourne male-only saunas. The low participation rate has highlighted the need to consider alternative methods for making contact with men in the saunas or offering incentives to participate in future screening programs. Although anonymous participation encouraged participation for some men, future programs should attempt to obtain contact details for follow up of positive test results.

Identification of a candidate membrane protein for the basolateral peptide transporter of rat small intestine.

A candidate protein for the basolateral peptide transporter of rat jejunum is described. Vascular perfusion of the photoaffinity label, [4-azido-D-phe]-L-ala (2.5mM), had no effect on the transepithelial transport of the non-hydrolysable dipeptide D-phe-L-gln (1mM) from the lumen, its mucosal accumulation or wash-out into the vascular perfusate. When the label was perfused luminally, the transepithelial transport of D-phe-L-gln was inhibited by 38% (P<0.001) and accumulation increased by 62% (P<0.05). These data are consistent with those of a basolateral transporter that is strongly asymmetric in its substrate binding and transport properties. Labelling of basolateral membrane vesicles with [4-azido-3,5-3H-D-phe]-L-ala revealed that the majority of label was incorporated into a single protein of M(r)112+/-2 kDa and pI 6.5. MALDI-TOF analysis of tryptic digests of the protein followed by database searches established that this protein was novel with no obvious similarity to PepT1, the apical membrane transporter.

Peptide mimics as substrates for the intestinal peptide transporter.

4-Aminophenylacetic acid (4-APAA), a peptide mimic lacking a peptide bond, has been shown to interact with a proton-coupled oligopeptide transporter using a number of different experimental approaches. In addition to inhibiting transport of labeled peptides, these studies show that 4-APAA is itself translocated. 4-APAA transport across the rat intact intestine was stimulated 18-fold by luminal acidification (to pH 6.8) as determined by high performance liquid chromatography (HPLC); in enterocytes isolated from mouse small intestine the intracellular pH was reduced on application of 4-APAA, as shown fluorimetrically with the pH indicator carboxy-SNARF; 4-APAA trans-stimulated radiolabeled peptide transport in brush-border membrane vesicles isolated from rat renal cortex; and in Xenopus oocytes expressing PepT1, 4-APAA produced trans-stimulation of radiolabeled peptide efflux, and as determined by HPLC, was a substrate for translocation by this transporter. These results with 4-APAA show for the first time that the presence of a peptide bond is not a requirement for rapid translocation through the proton-linked oligopeptide transporter (PepT1). Further investigation will be needed to determine the minimal structural requirements for a molecule to be a substrate for this transporter.

The influence of luminal pH on transport of neutral and charged dipeptides by rat small intestine, in vitro.

Four hydrolysis-resistant dipeptides (D-phenylalanyl-L-alanine, D-phenylalanyl-L-glutamine, D-phenylalanyl-L-glutamate and D-phenylalanyl-L-lysine) were synthesized to investigate the effects of net charge on transmural dipeptide transport by isolated jejunal loops of rat small intestine. At a luminal pH of 7.4 and a concentration of 1 mM the two dipeptides with a net charge of -1 and +1 were transported at substantially slower rates (18 +/- 1.3 and 8.4 +/- 1.3 nmol min(-1)(g dry wt.)(-1), respectively) than neutral D-phenylalanyl-L-alanine and D-phenylalanyl-L-glutamine (87 +/- 0.2 and 197 +/- 14 nmol min(-1)(g dry wt.)(-1), respectively). We investigated the effects of luminal pH on dipeptide transport by varying the NaHCO3 content of Krebs Ringer perfusate equilibrated with 95% 02/5% CO2. The pH changes did not affect water transport, but serosal glucose appearance increased significantly at pH 6.8. Transmural transport of D-phenylalanyl-L-alanine and D-phenylalanyl-L-glutamine at pH 6.8 was stimulated (P < 0.01) by 61% and 49%, respectively, whereas the lower pH increased the rate for negatively charged D-phenylalanyl-L-glutamate by 306% (P < 0.01) and decreased that for positively charged D-phenylalanyl-L-lysine by 46% (P < 0.05). Increasing luminal pH to 8.0 inhibited D-phenylalanyl-L-alanine transport by 60%, whereas D-phenylalanyl-L-lysine transport was 60% faster.

Dipeptide transport and hydrolysis in rat small intestine, in vitro.

A range of natural and mixed D-/L-stereoisomer phenylalanine dipeptides was used to investigate peptide uptake and hydrolysis by isolated rings of rat jejunum. Characterisation of dipeptide hydrolysis by the brush border fraction revealed apparent Km values in the 0.1-1.0 mM range which, except for the charged dipeptides, were significantly higher than those for hydrolysis by the cytosolic fraction. Uptake of L-/L-dipeptides into jejunal rings, which was followed by HPLC, was unaffected by the presence of peptidase inhibitors in the incubation medium although the absorbed peptides were completely hydrolysed in the cytosol; comparison of the effects of excess leucine on dipeptide uptake and on the uptake of the two constituent amino acids were also consistent with absorption of intact dipeptide followed by cytosolic hydrolysis. The uptake of hydrolysis-resistant mixed D-/L-dipeptides was also studied and confirmed that peptide uptake preceded hydrolysis; D-alanyl-L-phenylalanine accumulated within the rings to twice the medium concentration.